Methods and compositions for extending shelf life of plant products

ABSTRACT

The invention provides compositions and methods related to selective inhibition of PPO11 and use for improving shelf life of a plant or parts thereof. In accordance with the invention, for example, compositions for topical application to a plant or part thereof, are provided that can reduce browning of the plant or part thereof to extend shelf life.

BACKGROUND OF THE INVENTION

This application claims the priority of U.S. Provisional Appl. Ser. No.61/704,602, filed Sep. 24, 2012, the entire disclosure of which isincorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“SEMB014US_ST25,” which is 9 kilobytes as measured in Microsoft Windowsoperating system and was created on Sep. 24, 2013, is filedelectronically herewith and incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for improvingshelf life of plants and plant parts.

DESCRIPTION OF RELATED ART

Polyphenol oxidases (PPOs) are a group of copper-binding proteins,widely distributed phylogenetically from bacteria to mammals, thatcatalyze the oxidation of phenolics to quinones which produce brownpigments in wounded tissues. PPO has been implicated in the formation ofpigments, oxygen scavenging and defense mechanism against plantpathogens and herbivorous insects. The oxidation of phenolic substratesby PPO is thought to be the major cause of browning coloration of manyfruits and vegetables during ripening, handling, storage and processing.This problem is of considerable importance to the food industry as itaffects the nutritional quality and appearance, reduces the consumeracceptability and therefore also results in significant economic impact,both to the food producers and to the food processing industry.

SUMMARY OF THE INVENTION

In one aspect, the invention provides compositions for topicalapplication to a plant or part thereof, comprising an amount of aPolyphenol Oxidase 11 (PPO11) inhibitory compound effective to suppressexpression of a PPO11 gene or ortholog thereof, wherein expression ofthe PPO11 gene or ortholog thereof in the absence of the inhibitorycompound positively correlates with browning of the plant or partthereof. The composition may also comprise a transfer agent, a buffer,and/or an organosilicone preparation as transfer agent. The PPO11inhibitory compound may comprise, for example, a sense ssDNA, sensessRNA, dsRNA, dsDNA, a double stranded DNA/RNA hybrid, anti-sense ssDNA,or anti-sense ssRNA. In certain embodiments, the PPO11 inhibitorycompound may comprise an antisense oligonucleotide or dsRNA, or anucleic acid encoding an antisense oligonucleotide or dsRNA effective tosuppress expression of PPO11. In further embodiments, the PPO11inhibitory compound may comprise a polynucleotide molecule that is atleast 18 to about 24, about 25 to about 50, about 51 to about 100, about101 to about 300, about 301 to about 500, or at least about 500 or morenucleotides in length. In additional embodiments, the plant may belettuce and the PPO11 gene may be encoded by a sequence comprising SEQID NO:9. The PPO11 inhibitory compound may comprise a polynucleotidecomprising all or a part of a polynucleotide selected from the groupconsisting of SEQ ID NOs:1-9, or a complement thereof. In certainembodiments, the amount of PPO11 inhibitory compound is effective toreduce browning, or increase shelf life, of a processed plant product.

In another aspect, the invention provides methods for reducing browningor increasing shelf life of a plant, or part or product thereof,comprising topically applying to a surface of the plant or a partthereof a composition described herein. In certain embodiments, theplant is a potato, apple, spinach, or lettuce plant. In one embodiment,the plant is lettuce, and the PPO inhibitory compound comprises apolynucleotide comprising at least 21 contiguous nucleotides of SEQ IDNO:9, or a complement thereof.

In yet another aspect, the invention provides expression cassettescomprising a selected DNA operably linked to a heterologous promoter,the selected DNA encoding an antisense RNA, including dsRNA sequences,effective to suppress expression of a PPO11 gene or ortholog thereof,wherein expression of the PPO11 gene is positively correlated withbrowning or reduced shelf life of the plant, or part or product thereof.In certain embodiments, the promoter is a leaf-specific promoter. Invarious aspects, the selected DNA is operably linked to the promoter inantisense orientation; and/or the PPO11 gene encodes the polypeptideencoded by SEQ ID NO:9; the selected DNA comprises all or a portion of asequence selected from the group consisting of SEQ ID NOs:1-9. Incertain embodiments, expression of the selected DNA induces genesilencing, mRNA cleavage, or repressed translation of mRNA of a PPO11gene. In further embodiments, the selected DNA is at least 90% identicalto at least 200 contiguous nucleotides of SEQ ID NO:9 or a complementthereof; or comprises at least 18, 19, 20, 21, or 22 contiguousnucleotides of SEQ ID NO:334 or a complement thereof; and whereintranscription of the selected DNA suppresses expression of the PPO genethat is positively correlated with browning or reduced shelf life of theplant, or portion or product thereof. The invention also includesvectors comprising the expression cassette described above.

In still yet another aspect, the invention provides transgenic plantcomprising the above described expression cassette. In certainembodiments, the plant may be a dicot, a lettuce plant, an R0 transgenicplant, a progeny plant of any generation of an R0 transgenic plant,wherein the transgenic plant has inherited the selected DNA from the R0transgenic plant, and/or a progeny plant or a plant part derivedtherefrom that exhibits improved shelf life and reduced postharvestlosses.

In still yet another aspect, the invention provides seed of the abovedescribed transgenic plant, wherein the seed comprises the expressioncassette. In certain embodiments, the seed exhibits an improved shelflife and reduced postharvest losses. The invention also includestransgenic cells of the above described transgenic plant , wherein thecell comprises the expression cassette, a processed product of the plantor of a progeny thereof, wherein the processed product exhibits animproved shelf life, and the processed product, may be further definedas a head of lettuce, an apple fruit, a potato tuber, or a portion orproduct thereof.

In certain further aspects, the invention provides methods ofdown-regulating activity of a PPO11 gene in a plant, the methodscomprising introducing into the plant an expression cassette provided bythe invention; and selecting a plant with decreased PPO11 activitycompared to a plant in which the expression cassette has not beenintroduced. In certain embodiments, the PPO11 gene encodes mRNAcomprising SEQ ID NO:9, and the plant is lettuce. The selected DNA mayencode at least 21 continuous nucleotides of a PPO11 mRNA or acomplement thereof, effective to down regulate expression of PPO11; theselected DNA may be operably linked to the promoter in the antisenseorientation; and/or be in sense and antisense orientation. In certainembodiments, introducing the expression cassette comprises plantbreeding and/or genetic transformation. In further embodiments, thepolynucleotide may be an antisense or RNAi construct, the selected DNAencodes a ribozyme or zinc-finger protein that inhibits the expressionof PPO11. The plant may also be a lettuce, potato, apple, or spinachplant.

In still further aspects, the invention provides methods for makinghuman food comprising obtaining a plant according to the invention,growing the plant under plant growth conditions to produce plant tissuefrom the plant; and preparing food for human or animal consumption fromthe plant tissue.

In yet other aspects, the invention provides methods for identifyingplants having reduced expression or lacking expression of a PPO11 geneor ortholog thereof, comprising obtaining a plurality of plants, inwhich expression is positively correlated with browning or reduced shelflife of a processed product of the plant, selecting one or more screenedplants comprising decreased expression of the PPO11 gene or orthologthereof relative to expression of the PPO11 gene or ortholog thereof ina reference plant of the same crop species, a parent plant, or anotherwise isogenic plant, and which displays browning or reduced shelflife. In certain embodiments, the plurality of plants are obtained byrandom mutagenesis, are transgenic plants, comprise 10, 100, or 1000 ormore plants, and/or are varieties of the same species of plants. Incertain embodiments, screening for expression of the PPO gene comprisesdetermining an abundance of PPO11 RNA, determining PPO activity, ordetermining abundance of a protein or RNA encoded by SEQ ID NO:9. Incertain embodiment, the method further comprises crossing the one ormore plants with reduced expression of PPO11 to a different plant.

In still yet another aspect, the invention provides methods foridentifying a polymorphism genetically linked to a PPO11 gene orortholog thereof, comprising: obtaining DNA of a population of plantswherein members of the population vary for expression of PPO11; andidentifying at least a first polymorphism in said population that isassociated with a reduced expression of PPO11 relative to members of thepopulation that do not comprise said polymorphism.

In still yet another aspect, the invention provides methods of lettucebreeding comprising assaying lettuce plants, or seeds that produce thelettuce plants, for the presence of at least a first genetic markergenetically linked to a chromosomal region conferring reduced PPO11expression relative to a plant lacking the region, wherein the region isa low PPO11 expression contributing QTL between 185700k and 185800k onlettuce chromosome 4; and selecting at least a first lettuce plant orseed that produces the plant comprising the genetic marker and the QTLthat confers reduced expression of PPO11; and crossing the first plantwith itself or a second plant to produce progeny plants comprising theQTL that confers reduced expression of PPO.

In still yet another aspect, the invention provides methods ofidentifying a lettuce plant that displays reduced browning or increasedshelf life comprising: detecting in a first lettuce plant at least oneallele of a marker that is associated with reduced browning or increasedshelf life, wherein the marker is genetically linked to a region between185700k and 185800k on lettuce chromosome 4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Eleven putative PPO genes exist in lettuce. Based on analysis ofnucleotide substitution per 100 nucleotide residues, the relative levelof homology among the PPO genes is shown.

FIG. 2: Transcript abundances of the 11 lettuce PPO genes were assayedat 0, 4, 8, 12, 24, 36, 48, 72, and 96 hours after harvest to identifywhich PPO genes correlate in expression to the incidence of browning inuntreated lettuce ribs. Surprisingly, despite high homology among PPOgenes, PPO11 was identified as the transcript whose expressioncorrelated with and predicted the visual browning phenotype.

FIGS. 3A-B: FIG. 3A depicts a strong correlation between PPO11expression levels 9 days after processing and cold storage with visualdiscoloration ratings for two crisphead romaine cross and three icebergvarieties grown in San Juan Bautista. Each point represents the leastsquare mean for a single cultivar with six biological replicates forboth gene expression and visual ratings. FIG. 3B is a validationexperiment with 10 cultivars grown in Yuma, AZ again demonstrating astatistically significant correlation between PPO11 expression levels 9days after processing and cold storage with visual discoloration ratingsfor two crisphead romaine cross, three iceberg, and five Romainecultivars. Each point represents the least square mean for a singlecultivar with six biological replicates for both gene expression andvisual ratings. Iceberg “ICE”, crisphead-romaine cross “CRC,” andromaine “ROM” lettuce.

FIG. 4: Plot of Visual Discoloration vs PAL1, T₀; vs PPO2, T₀; vs PPO11,T₉; vs PPO8, T₀. RQ PAL 1, T₀=relative quantification to ubiquitin (RQ)for phenylalanine lyase homolog 1 (PAL1) , at time 0 (T₀). Thiscorrelation matrix produced in JMPv10 shows the individual variable,i.e. average visual browning scores on the vertical axis in the row thevariable is listed and on the horizontal axis in the column it islisted. Plotted are the most correlated gene expression at harvest or 9days after processing. PPO11 shows the largest range of expression (˜200to ˜11000).

FIG. 5: Depicted is a histogram of total PPO activity (uM*min-1*ug-1conversion of catechol) for a population of 23 Romaine cultivars asassessed from central midrib tissue at time of processing. Counts on thevertical axis are the number of cultivars with a U PPO activity in theindicated range. This shows from a selection of elite Romaine cultivarsthat there is 3-fold range in total PPO activity in the central midribtissue. Sub-selections of the highest and lowest PPO activity cultivarsfrom this experiment were chosen to phenotype for visualdiscoloration/shelf life.

FIG. 6: PPO Enzymatic Activity Predictions of visual Discoloration.Depicted is a bar chart of the visual discoloration for a subset oflines previously selected to have the highest or lowest U PPO activityfrom a selection of 23 Romaine as described (see FIG. 5). Larger numberson the vertical axis indicated more discoloration. The coded cultivarsare grouped by previously determined high or low PPO activity. Theactivity level was not predictive of shelf life, as two of the highestenzymatic activity lines were the most resistant in discolorationphenotype.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1—PPO11_Trigger1 sequence (189nt).

SEQ ID NO:2—PPO11_Trigger2 (225nt).

SEQ ID NO:3—PPO11_Trigger3 (225nt).

SEQ ID NO:4—PPO11_Trigger4 (226nt).

SEQ ID NO:5—PPO11_antisense ssDNA (25nt).

SEQ ID NO:6—PPO11_antisense ssDNA (25nt).

SEQ ID NO:7—PPO11_dsRNA (25nt).

SEQ ID NO:8—PPO11_dsRNA (25nt).

SEQ ID NO:9—Lactuca sativa (lettuce) deduced PPO11 mRNA (1922nt).

SEQ ID NO:10—PPO11 flanking sequence (SequenceChr4:185719000-185720000).

SEQ ID NO:11—PPO11 flanking sequence (SequenceChr4:1185723000-185724000).

SEQ ID NOs:12 -34—Gene-specific PCR primers.

DETAILED DESCRIPTION OF THE INVENTION

Polyphenol oxidases (PPOs) are a group of copper-binding proteins,widely distributed phylogenetically from bacteria to mammals thatcatalyze the oxidation of phenolics to quinones and produce brownpigments in wounded tissues. The present invention overcomes thelimitations of the prior art by providing novel methods, compositions,and plants for extending shelf life of plant products by specificreduction of expression of a PPO that positively correlates withbrowning in plants. PPO11 of lettuce provides a non-limiting example ofa PPO that correlates with browning in lettuce. It was recognized thatexpression of PPO11 increases over time after harvest and correlateswith reduced shelf life and increased browning. Compositions aredisclosed that comprise selective PPO inhibitory compounds to inhibit aspecific PPO that positively correlates with browning such as PPO11, andmethods to reduce browning or increase shelf life. Also disclosed areexpression cassettes and vectors comprising a selected DNA whoseexpression is effective to suppress expression of PPO11, reducebrowning, and/or increase shelf life of plants or plant parts.Transgenic plants or plants comprising PPO11 inhibitory compounds andmethods of producing such are also disclosed. Plants or plant varietiesthat harbor a PPO gene or promoter mutations that result in reduced PPOgene expression and methods of assaying PPO gene expression as a proxyfor browning are also disclosed herein. In particular embodiments, thePPO gene that positively correlates with browning is a PPO11 gene fromlettuce and/or the corresponding ortholog from apple or potato. Inaddition, methods of producing PPO genes with reduced expression areprovided.

Definitions

In the description and tables herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided. Unlessotherwise noted, terms are to be understood according to conventionalusage by those of ordinary skill in the relevant art.

Where a term is provided in the singular, the inventors also contemplateaspects of the invention described by the plural of that term.

As used herein, the term “selective PPO inhibitory compound” refers to acompound that suppresses or reduces an activity of PPO enzymaticactivity, or expression of PPO, such as for example synthesis of mRNAencoding a PPO polypeptide (transcription) and/or synthesis of a PPOpolypeptide from PPO mRNA (translation). In some embodiments theselective PPO inhibitory compound specifically inhibits a PPO thatpositively correlates with browning of the plant. In certainembodiments, the PPO inhibitory compound is an inhibitor of PPO11 or thecorresponding gene in apple or potatoes.

As used herein, a PPO that positively correlates with browning of theplant refers to a specific PPO homolog that exhibits an increase ofexpression after harvest when the browning/discoloration increases oroccurs. In certain embodiments, the increase of expression is observedduring the period of 36 hrs after harvest. The PPO that positivelycorrelates with browning of the plant (plant surface or parts of aplant) also exhibits an increase of expression that correlates withvisual discoloration of the plant surface. In certain embodiments, suchPPO is PPO11. In certain embodiments, the PPO is a homolog of PPO11 oflettuce correlated with the presence of browning, such as from apple orpotato, which may therefore be targeted for reduction of browning.

As used herein, the term “expression cassette” refers to a DNA sequencethat comprises a selected DNA to be transcribed. In addition, theexpression cassette comprises at least all DNA elements required forexpression. After successful transformation, the expression cassettedirects the cell's machinery to make RNA. In certain embodiments, theexpression cassette is an iRNA or siRNA expression cassette thatsuppresses expression of a PPO that positively correlates with browningof a plant.

Different expression cassettes can be transformed into differentorganisms including bacteria, yeast, plants, and mammalian cells as longas the correct regulatory sequences are used.

As used herein, the term “abundance of a protein” refers to the amountof the specific protein relative to the amount of total protein orrelative to the weight or volume of the cell, tissue, plant, or plantpart tested.

As used herein, the term “abundance of a mRNA” refers to the amount ofthe specific mRNA relative to the amount of total protein or relative tothe weight or volume of the cell, tissue, plant, or plant part tested.

As used herein, the term “expression” refers to the combination ofintracellular processes, including transcription and translationundergone by a coding DNA molecule such as a structural gene to producea polypeptide.

As used herein, the term “genetic transformation” refers to process ofintroducing a DNA sequence or construct (e.g., a vector or expressioncassette) into a cell or protoplast in which that exogenous DNA isincorporated into a chromosome or is capable of autonomous replication.

As used herein, the term “heterologous” refers to a sequence which isnot normally present in a given host genome in the genetic context inwhich the sequence is currently found In this respect, the sequence maybe native to the host genome, but be rearranged with respect to othergenetic sequences within the host sequence. For example, a regulatorysequence may be heterologous in that it is linked to a different codingsequence relative to the native regulatory sequence.

As used herein, the term “obtaining” when used in conjunction with atransgenic plant cell or transgenic plant, means either for 1)transforming a non-transgenic plant cell or plant to create thetransgenic plant cell or plant, or for 2) planting transgenic plant seedto produce the transgenic plant cell or plant. Such a transgenic plantseed may be from an R₀ transgenic plant or may be from a progeny of anygeneration thereof that inherits a given transgenic sequence from astarting transgenic parent plant.

As used herein, the term “promoter” refers to a recognition site on aDNA sequence or group of DNA sequences that provides an expressioncontrol element for a structural gene and to which RNA polymerasespecifically binds and initiates RNA synthesis (transcription) of thatgene

As used herein, the term “R₀ transgenic plant” refers to a plant thathas been genetically transformed or has been regenerated from a plantcell or cells that have been genetically transformed.

As used herein, the term “regeneration” refers to the process of growinga plant from a plant cell (e.g., plant protoplast, callus or explant).

As used herein, the term “transformation construct” refers to a chimericDNA molecule which is designed for introduction into a host genome bygenetic transformation. Preferred transformation constructs willcomprise all of the genetic elements necessary to direct the expressionof one or more exogenous genes. In particular embodiments of the instantinvention, it may be desirable to introduce a transformation constructinto a host cell in the form of an expression cassette.

As used herein, the term “transformed cell” refers to a cell the DNAcomplement of which has been altered by the introduction of an exogenousDNA molecule into that cell.

As used herein, the term “transgene” refers to a segment of DNA whichhas been incorporated into a host genome or is capable of autonomousreplication in a host cell and is capable of causing the expression ofone or more coding sequences. Exemplary transgenes will provide the hostcell, or plants regenerated therefrom, with a novel phenotype relativeto the corresponding non-transformed cell or plant. Transgenes may bedirectly introduced into a plant by genetic transformation, or may beinherited from a plant of any previous generation which was transformedwith the DNA segment.

As used herein, the term “transgenic plant” refers to a plant or progenyplant of any subsequent generation derived therefrom, wherein the DNA ofthe plant or progeny thereof contains an introduced exogenous DNAsegment not naturally present in a non-transgenic plant of the samestrain. The transgenic plant may additionally contain sequences whichare native to the plant being transformed, but wherein the “exogenous”gene has been altered in order to alter the level or pattern ofexpression of the gene, for example, by use of one or more heterologousregulatory or other elements.

As used herein, the term “vector” refers to a DNA molecule designed fortransformation into a host cell. Some vectors may be capable ofreplication in a host cell. A plasmid is an exemplary vector, as areexpression cassettes isolated therefrom.

As used herein, the terms “DNA,” “DNA molecule,” and “DNA polynucleotidemolecule” refer to a single-stranded DNA or double-stranded DNA moleculeof genomic or synthetic origin, such as, a polymer ofdeoxyribonucleotide bases or a DNA polynucleotide molecule.

As used herein, the terms “DNA sequence,” “DNA nucleotide sequence,” and“DNA polynucleotide sequence” refer to the nucleotide sequence of a DNAmolecule. ssDNA refers to single-stranded DNA; dsDNA refers todouble-stranded DNA.

As used herein, the term “gene” refers to any portion of a nucleic acidthat provides for expression of a transcript or encodes a transcript. A“gene” thus includes, but is not limited to, a promoter region, 5′untranslated regions, transcript encoding regions that can includeintronic regions, and 3′ untranslated regions.

As used herein, the terms “RNA,” “RNA molecule,” and “RNA polynucleotidemolecule” refer to a single-stranded RNA or double-stranded RNA moleculeof genomic or synthetic origin, such as, a polymer of ribonucleotidebases that comprise single or double stranded regions. ssRNAspecifically refers to single-stranded RNA; ds refers to double-strandedRNA.

Unless otherwise stated, nucleotide sequences in the text of thisspecification are given, when read from left to right, in the 5′ to 3′direction. The nomenclature used herein is that required by Title 37 ofthe United States Code of Federal Regulations § 1.822 and set forth inthe tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.

As used herein, a “plant surface” refers to any exterior portion of aplant. Plant surfaces thus include, but are not limited to, the surfacesof flowers, stems, tubers, fruit, anthers, pollen, leaves, roots, orseeds. A plant surface can be on a portion of a plant that is attachedto other portions of a plant or on a portion of a plant that is detachedfrom the plant.

As used herein, the phrase “polynucleotide is not operably linked to apromoter” refers to a polynucleotide that is not covalently linked to apolynucleotide promoter sequence that is specifically recognized byeither a DNA dependent RNA polymerase II protein or by a viral RNAdependent RNA polymerase in such a manner that the polynucleotide willbe transcribed by the DNA dependent RNA polymerase II protein or viralRNA dependent RNA polymerase. A polynucleotide that is not operablylinked to a promoter can be transcribed by a plant RNA dependent RNApolymerase.

As used herein, a polynucleotides may be in the form of ssDNA, encompassdsDNA equivalents, dsRNA equivalents, ssRNA equivalents, ssRNAcomplements, ssDNA as shown, and ssDNA complements.

As used herein, a first nucleic-acid sequence, selected DNA, orpolynucleotide is “operably” connected or “linked” with a second nucleicacid sequence when the first nucleic acid sequence is placed in afunctional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to an RNA and/or protein-codingsequence, or a sequence encoding an iRNA, an siRNA, or a nucleic acidencoding an antisense oligonucleotide if the promoter provides fortranscription or expression of the RNA or coding sequence. Generally,operably linked DNA sequences are contiguous and, where necessary tojoin two protein-coding regions, are in the same reading frame. SelectedDNA refers to a DNA segment which one desires to introduce or hasintroduced into a plant genome by genetic transformation

In certain embodiment, the selected DNA is an antisense or RNAiconstruct. In another embodiment, the selected DNA encodes a ribozyme,or zinc-finger protein.

As used herein, the phrase “organosilicone preparation” refers to aliquid comprising one or more organosilicone compounds, wherein theliquid or components contained therein, when combined with apolynucleotide in a composition that is topically applied to a targetplant surface, enable the polynucleotide to enter a plant cell.Exemplary organosilicone preparations include, but are not limited to,preparations marketed under the trade names “Silwet®” or “ BREAK-THRU®”and preparations provided in the following Table. In certainembodiments, an organosilicone preparation can enable a polynucleotideto enter a plant cell in a manner permitting a polynucleotide mediatedsuppression of target gene expression in the plant cell.

As used herein, the phrases “reduced browning,” “increased shelf life,”“improved shelf life,” “reduced postharvest losses,” or “improving shelflife and reducing postharvest losses” refer to any measurable increasein shelf life, reduction in browning, or reduction in postharvest lossobserved in a plant or part thereof subjected to the present inventionwhen compared to the plant or part thereof not subjected to the presentinvention. In certain embodiments, an increase in shelf life orreduction in postharvest loss in a plant or plant part can be determinedin a comparison to a control plant or plant part that has not beentreated with a composition comprising a polynucleotide and a transferagent. When used in this context, a control plant is a plant that hasnot undergone treatment with a PPO inhibitory compound or apolynucleotide, non-transgenic plants, or plants not exhibiting reducedexpression of a PPO. Such control plants would include, but are notlimited to, untreated plants or mock treated plants. In certainembodiments, the phrases relate to non-transgenic plants or plants thatdo not comprise an expression cassette that effectively suppresses PPOactivity.

As used herein, the phrase “provides for a reduction,” “effective tosuppress,” and “effectively suppresses” when used in the context of atranscript or a protein in a plant or plant part, refers to anymeasurable decrease in the level of transcript or protein in a plant orplant part. Thus, expression of a gene can be suppressed when there is areduction in levels of a transcript from the gene, a reduction in levelsof a protein encoded by the gene, a reduction in the activity of thetranscript from the gene, a reduction in the activity of a proteinencoded by the gene, any one of the preceding conditions, or anycombination of the preceding conditions. In this context, the activityof a transcript includes, but is not limited to, its ability to betranslated into a protein and/or to exert any RNA-mediated biologic orbiochemical effect. In this context, the activity of a protein includes,but is not limited to, its ability to exert any protein-mediatedbiologic or biochemical effect. In certain embodiments, a suppression ofgene expression in a plant or plant part can be determined in acomparison of gene product levels or activities in a treated plant to acontrol plant or plant part that has not been treated with a compositioncomprising a polynucleotide and a transfer agent. When used in thiscontext, a control plant or plant part is a plant or plant part that hasnot undergone treatment with polynucleotide and a transfer agent. Suchcontrol plants or plant parts would include, but are not limited to,untreated or mock treated plants and plant parts. In certainembodiments, a reduction of the level of a transcript or protein in aplant or plant part can be determined in a comparison to a control plantor plant part that has not been treated with a composition comprising apolynucleotide and a transfer agent. When used in this context, acontrol plant or plant part is a plant or plant part that has notundergone treatment with a PPO inhibitory compound, a polynucleotide.Such control plants or plant parts would include, but are not limitedto, untreated or mock treated plants and plant parts. In certainembodiments, the phrases relate to non-transgenic plants or plants thatdo not comprise an expression cassette that having the continuousnucleotides of a PPO gene, or a plant that does not comprise atransgene.

As used herein, the phrase “wherein said plant does not comprise atransgene” refers to a plant that lacks either a DNA molecule comprisinga promoter that is operably linked to a polynucleotide or a recombinantviral vector.

As used herein, the term “transcript” corresponds to any RNA that isproduced from a gene by the process of transcription. A transcript of agene can thus comprise a primary transcription product which can containintrons or can comprise a mature RNA that lacks introns.

As used herein, “polynucleotide” may refer to a DNA or RNA moleculecontaining multiple nucleotides and generally refers both to“oligonucleotides” (a polynucleotide molecule of 18-25 nucleotides inlength) and longer polynucleotides of 26 or more nucleotides.Embodiments of this invention include compositions includingoligonucleotides having a length of 18-25 nucleotides (18-mers, 19-mers,20-mers, 21-mers, 22-mers, 23-mers, 24-mers, or 25-mers), and longer, ormedium-length polynucleotides having a length of 26 or more nucleotides(polynucleotides of 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about90, about 95, about 100, about 110, about 120, about 130, about 140,about 150, about 160, about 170, about 180, about 190, about 200, about210, about 220, about 230, about 240, about 250, about 260, about 270,about 280, about 290, or about 300 nucleotides), or long polynucleotideshaving a length greater than about 300 nucleotides (e.g.,polynucleotides of between about 300 to about 400 nucleotides, betweenabout 400 to about 500 nucleotides, between about 500 to about 600nucleotides, between about 600 to about 700 nucleotides, between about700 to about 800 nucleotides, between about 800 to about 900nucleotides, between about 900 to about 1000 nucleotides, between about300 to about 500 nucleotides, between about 300 to about 600nucleotides, between about 300 to about 700 nucleotides, between about300 to about 800 nucleotides, between about 300 to about 900nucleotides, or about 1000 nucleotides in length, or even greater thanabout 1000 nucleotides in length, for example up to the entire length ofa target Polyphenol oxidase (PPO) gene, such as PPO11 including codingor non-coding or both coding and non-coding portions of the targetPolyphenol oxidase (PPO) gene). Where a polynucleotide isdouble-stranded, its length can be similarly described in terms of basepairs.

Polynucleotide compositions used in the various embodiments of thisinvention include compositions including oligonucleotides,polynucleotides, or a mixture of both, including: RNA or DNA or RNA/DNAhybrids or chemically modified oligonucleotides or polynucleotides or amixture thereof. In certain embodiments, the polynucleotide may be acombination of ribonucleotides and deoxyribonucleotides, for example,synthetic polynucleotides consisting mainly of ribonucleotides but withone or more terminal deoxyribonucleotides or synthetic polynucleotidesconsisting mainly of deoxyribonucleotides but with one or more terminaldideoxyribonucleotides. In certain embodiments, the polynucleotideincludes non-canonical nucleotides such as inosine, thiouridine, orpseudouridine. In certain embodiments, the polynucleotide includeschemically modified nucleotides. Examples of chemically modifiedoligonucleotides or polynucleotides are well known in the art; see, forexample, U.S. Patent Publication 2011/0171287, U.S. Patent Publication2011/0171176, U.S. Patent Publication 2011/0152353, U.S. PatentPublication 2011/0152346, and U.S. Patent Publication 2011/0160082,which are herein incorporated by reference. Illustrative examplesinclude, but are not limited to, the naturally occurring phosphodiesterbackbone of an oligonucleotide or polynucleotide which can be partiallyor completely modified with phosphorothioate, phosphorodithioate, ormethylphosphonate internucleotide linkage modifications, modifiednucleoside bases or modified sugars can be used in oligonucleotide orpolynucleotide synthesis, and oligonucleotides or polynucleotides can belabeled with a fluorescent moiety (e.g., fluorescein or rhodamine) orother label (e.g., biotin).

Polynucleotides can be single- or double-stranded RNA, single- ordouble-stranded DNA, double-stranded DNA/RNA hybrids, and modifiedanalogues thereof. In certain embodiments of the invention, thepolynucleotides that provide single-stranded RNA in the plant cell maybe: (a) a single-stranded RNA molecule (ssRNA), (b) a single-strandedRNA molecule that self-hybridizes to form a double-stranded RNAmolecule, (c) a double-stranded RNA molecule (dsRNA), (d) asingle-stranded DNA molecule (ssDNA), (e) a single-stranded DNA moleculethat self-hybridizes to form a double-stranded DNA molecule, (f) asingle-stranded DNA molecule including a modified Pol III gene that istranscribed to an RNA molecule, (g) a double-stranded DNA molecule(dsDNA), (h) a double-stranded DNA molecule including a modified Pol IIIgene that is transcribed to an RNA molecule, and (i) a double-stranded,hybridized RNA/DNA molecule, or combinations thereof. In certainembodiments, these polynucleotides can comprise both ribonucleic acidresidues and deoxyribonucleic acid residues. In certain embodiments,these polynucleotides include chemically modified nucleotides ornon-canonical nucleotides. In certain embodiments of the methods, thepolynucleotides include double-stranded DNA formed by intramolecularhybridization, double-stranded DNA formed by intermolecularhybridization, double-stranded RNA formed by intramolecularhybridization, or double-stranded RNA formed by intermolecularhybridization. In certain embodiments where the polynucleotide is adsRNA, the anti-sense strand will comprise at least 18 nucleotides thatare essentially complementary to the target Polyphenol oxidase (PPO)gene. In certain embodiments the polynucleotides include single-strandedDNA or single-stranded RNA that self-hybridizes to form a hairpinstructure having an at least partially double-stranded structureincluding at least one segment that will hybridize to RNA transcribedfrom the gene targeted for suppression. Not intending to be bound by anymechanism, it is believed that such polynucleotides are or will producesingle-stranded RNA with at least one segment that will hybridize to RNAtranscribed from the gene targeted for suppression. In certainembodiments, the polynucleotides can be operably linked to apromoter—generally a promoter functional in a plant, for example, a polII promoter, a pol III promoter, a pol IV promoter, or a pol V promoter.

The polynucleotide molecules of the present invention are designed tomodulate expression by inducing regulation or suppression of anendogenous Polyphenol oxidase (PPO) gene, such as PPO11, to reduceexpression of PPO11 in a plant and are designed to have a nucleotidesequence essentially identical or essentially complementary to thenucleotide sequence of an endogenous Polyphenol oxidase (PPO) gene of aplant or to the sequence of RNA transcribed from an endogenousPolyphenol oxidase (PPO) gene of a plant, which can be coding sequenceor non-coding sequence. These effective polynucleotide molecules thatmodulate expression are referred to herein as “a trigger, or triggers”.By “essentially identical” or “essentially complementary” it is meantthat the trigger polynucleotides (or at least one strand of adouble-stranded polynucleotide) have sufficient identity orcomplementarity to the endogenous gene or to the RNA transcribed fromthe endogenous Polyphenol oxidase (PPO) gene (e.g., the transcript) tosuppress expression of the endogenous Polyphenol oxidase (PPO) gene(e.g., to effect a reduction in levels or activity of the genetranscript and/or encoded protein). In certain embodiments, the triggerpolynucleotides provided herein can be directed to a Polyphenol oxidase(PPO) transgene present in the plant. Polynucleotides of the methods andcompositions provided herein need not have 100 percent identity to acomplementarity to the endogenous Polyphenol oxidase (PPO) gene or tothe RNA transcribed from the endogenous Polyphenol oxidase (PPO) gene(i.e., the transcript) to suppress expression of the endogenousPolyphenol oxidase (PPO) gene (i.e., to effect a reduction in levels oractivity of the gene transcript or encoded protein). Thus, in certainembodiments, the polynucleotide or a portion thereof is designed to beessentially identical to, or essentially complementary to, a sequence ofat least 18 or 19 contiguous nucleotides in either the target gene ormessenger RNA transcribed from the target gene (e.g., the transcript).In certain embodiments, an “essentially identical” polynucleotide has100 percent sequence identity or at least about 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identitywhen compared to the sequence of 18 or more contiguous nucleotides ineither the endogenous target gene or to an RNA transcribed from thetarget gene (e.g., the transcript). In certain embodiments, an“essentially complementary” polynucleotide has 100 percent sequencecomplementarity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, or 99 percent sequence complementarity whencompared to the sequence of 18 or more contiguous nucleotides in eitherthe target gene or RNA transcribed from the target gene.

In certain embodiments, polynucleotides used in the methods andcompositions provided herein can be essentially identical or essentiallycomplementary to any of: i) conserved regions of Polyphenol oxidase(PPO) genes of both monocot and dicot plants; ii) conserved regions ofPolyphenol oxidase (PPO) genes of monocot plants; or iii) conservedregions of Polyphenol oxidase (PPO) genes of dicot plants. Suchpolynucleotides that are essentially identical or essentiallycomplementary to such conserved regions can be used to improve shelflife and reduce postharvest losses by suppressing expression ofPolyphenol oxidase (PPO) genes in various dicot and/or monocot plants.

Polynucleotides containing mismatches to the target gene or transcriptcan thus be used in certain embodiments of the compositions and methodsprovided herein. In certain embodiments, a polynucleotide can compriseat least 19 contiguous nucleotides that are essentially identical oressentially complementary to said gene or said transcript or comprisesat least 19 contiguous nucleotides that are essentially identical oressentially complementary to the target gene or target gene transcript.In certain embodiments, a polynucleotide of 19 continuous nucleotidesthat is essentially identical or essentially complementary to theendogenous target gene or to a RNA transcribed from the target gene(e.g., the transcript) can have 1 or 2 mismatches to the target gene ortranscript. In certain embodiments, a polynucleotide of 20 or morenucleotides that contains a contiguous 19 nucleotide span of identity orcomplementarity to the endogenous target gene or to an RNA transcribedfrom the target gene can have 1 or 2 mismatches to the target gene ortranscript. In certain embodiments, a polynucleotide of 21 continuousnucleotides that is essentially identical or essentially complementaryto the endogenous target gene or to an RNA transcribed from the targetgene (e.g., the transcript) can have 1, 2, or 3 mismatches to the targetgene or transcript. In certain embodiments, a polynucleotide of 22 ormore nucleotides that contains a contiguous 21 nucleotide span ofidentity or complementarity to the endogenous target gene or to an RNAtranscribed from the target gene can have 1, 2, or 3 mismatches to thetarget gene or transcript. In designing polynucleotides with mismatchesto an endogenous target gene or to an RNA transcribed from the targetgene, mismatches of certain types and at certain positions that are morelikely to be tolerated can be used. In certain exemplary embodiments,mismatches formed between adenine and cytosine or guanosine and uracilresidues are used as described by Du et al. Nucleic Acids Research,2005, Vol. 33, No. 5 1671-1677. In certain exemplary embodiments,mismatches in 19 base pair overlap regions can be at the low tolerancepositions 5, 7, 8 or 11 (from the 5′ end of a 19 nucleotide target) withwell tolerated nucleotide mismatch residues, at medium tolerancepositions 3, 4, and 12-17, and/or at the high tolerance nucleotidepositions at either end of the region of complementarity (i.e.,positions 1, 2, 18, and 19) as described by Du et al. Nucleic AcidsResearch, 2005, Vol. 33, No. 5 1671-1677. It is further anticipated thattolerated mismatches can be empirically determined in assays where thepolynucleotide is applied to the plants via the methods provided hereinand the treated plants assayed for suppression of Polyphenol oxidase(PPO) gene expression or appearance of improved shelf life and reducedpostharvest losses.

In certain embodiments, polynucleotide molecules are designed to have100 percent sequence identity with or complementarity to one allele orone family member of a given target Polyphenol oxidase (PPO) gene codingor non-coding sequence. Target Polyphenol oxidase (PPO) genes includeboth the Polyphenol oxidase (PPO) genes of SEQ ID NO:1-23 as well asorthologous Polyphenol oxidase (PPO) genes obtainable from other crops.In other embodiments, the polynucleotide molecules are designed to have100 percent sequence identity with or complementarity to multiplealleles or family members of a given target gene.

In certain embodiments, polynucleotide compositions and methods providedherein typically effect regulation or modulation (e. g., suppression) ofgene expression during a period during the life of the treated plant ofat least 1 week or longer and typically in systemic fashion. Forinstance, within days of treating a plant leaf with a polynucleotidecomposition of this invention, primary and transitive siRNAs can bedetected in other leaves lateral to and above the treated leaf and inapical tissue. In certain embodiments, methods of systemicallysuppressing expression of a gene in a plant, the methods comprisingtreating said plant with a composition comprising at least onepolynucleotide and a transfer agent, wherein said polynucleotidecomprises at least 18 or at least 19 contiguous nucleotides that areessentially identical or essentially complementary to a gene or atranscript encoding a Polyphenol oxidase (PPO) gene of the plant areprovided, whereby expression of the gene in said plant or progenythereof is systemically suppressed in comparison to a control plant thathas not been treated with the composition.

Compositions used to suppress a target gene can comprise one or morepolynucleotides that are essentially identical or essentiallycomplementary to multiple genes, or to multiple segments of one or moregenes. In certain embodiments, compositions used to suppress a targetgene can comprise one or more polynucleotides that are essentiallyidentical or essentially complementary to multiple consecutive segmentsof a target gene, multiple non-consecutive segments of a target gene,multiple alleles of a target gene, or multiple target genes from one ormore species.

In certain embodiments, the polynucleotide includes two or more copiesof a nucleotide sequence (of 18 or more nucleotides) where the copiesare arranged in tandem fashion. In another embodiment, thepolynucleotide includes two or more copies of a nucleotide sequence (of18 or more nucleotides) where the copies are arranged in inverted repeatfashion (forming an at least partially self-complementary strand). Thepolynucleotide can include both tandem and inverted-repeat copies.Whether arranged in tandem or inverted repeat fashion, each copy can bedirectly contiguous to the next, or pairs of copies can be separated byan optional spacer of one or more nucleotides. The optional spacer canbe unrelated sequence (i.e., not essentially identical to or essentiallycomplementary to the copies, nor essentially identical to, oressentially complementary to, a sequence of 18 or more contiguousnucleotides of the endogenous target gene or RNA transcribed from theendogenous target gene). Alternatively the optional spacer can includesequence that is complementary to a segment of the endogenous targetgene adjacent to the segment that is targeted by the copies. In certainembodiments, the polynucleotide includes two copies of a nucleotidesequence of between about 20 to about 30 nucleotides, where the twocopies are separated by a spacer no longer than the length of thenucleotide sequence.

Tiling

Polynucleotide trigger molecules can be identified by “tiling” genetargets in random length fragments, e.g., 200-300 polynucleotides inlength, with partially overlapping regions, e.g., 25 or so nucleotideoverlapping regions along the length of the target gene. Multiple genetarget sequences can be aligned and polynucleotide sequence regions withhomology in common are identified as potential trigger molecules formultiple targets. Multiple target sequences can be aligned and sequenceregions with poor homology are identified as potential trigger moleculesfor selectively distinguishing targets. To selectively suppress a singlegene, trigger sequences may be chosen from regions that are unique tothe target gene either from the transcribed region or the non-codingregions, e.g., promoter regions, 3′ untranslated regions, introns andthe like.

Polynucleotide fragments are designed along the length of the fulllength coding and untranslated regions of a Polyphenol oxidase (PPO)gene or family member as contiguous overlapping fragments of 200-300polynucleotides in length or fragment lengths representing a percentageof the target Polyphenol oxidase (PPO) gene. These fragments are appliedtopically (as sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA) todetermine the relative effectiveness in providing the improved shelflife and reduced postharvest losses phenotype. Fragments providing thedesired activity may be further subdivided into 50-60 polynucleotidefragments which are evaluated for providing the improved shelf life andreduced postharvest losses phenotype. The 50-60 base fragments with thedesired activity may then be further subdivided into 19-30 basefragments which are evaluated for providing the improved shelf life andreduced postharvest losses phenotype. Once relative effectiveness isdetermined, the fragments are utilized singly, or in combination in oneor more pools to determine effective trigger composition or mixture oftrigger polynucleotides for providing the improved shelf life andreduced postharvest losses phenotype.

Coding and/or non-coding sequences of Polyphenol oxidase (PPO) genefamilies in the crop of interest are aligned and 200-300 polynucleotidefragments from the least homologous regions amongst the alignedsequences are evaluated using topically applied polynucleotides (assense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA) to determine theirrelative effectiveness in providing the improved shelf life and reducedpostharvest losses phenotype. The effective segments are furthersubdivided into 50-60 polynucleotide fragments, prioritized by leasthomology, and reevaluated using topically applied polynucleotides. Theeffective 50-60 polynucleotide fragments are subdivided into 19-30polynucleotide fragments, prioritized by least homology, and againevaluated for induction of the improved shelf life and reducedpostharvest losses phenotype. Once relative effectiveness is determined,the fragments are utilized singly, or again evaluated in combinationwith one or more other fragments to determine the trigger composition ormixture of trigger polynucleotides for providing the yield/qualityphenotype.

Coding and/or non-coding sequences of Polyphenol oxidase (PPO) genefamilies in the crop of interest are aligned and 200-300 polynucleotidefragments from the most homologous regions amongst the aligned sequencesare evaluated using topically applied polynucleotides (as sense oranti-sense ssDNA or ssRNA, dsRNA, or dsDNA) to determine their relativeeffectiveness in inducing the improved shelf life and reducedpostharvest losses phenotype. The effective segments are subdivided into50-60 polynucleotide fragments, prioritized by most homology, andreevaluated using topically applied polynucleotides. The effective 50-60polynucleotide fragments are subdivided into 19-30 polynucleotidefragments, prioritized by most homology, and again evaluated forinduction of the improved shelf life and reduced postharvest lossesphenotype. Once relative effectiveness is determined, the fragments maybe utilized singly, or in combination with one or more other fragmentsto determine the trigger composition or mixture of triggerpolynucleotides for providing the improved shelf life and reducedpostharvest losses phenotype.

Also, provided herein are methods for identifying a preferredpolynucleotide for providing improved shelf life and reduced postharvestlosses in a plant. Populations of candidate polynucleotides that areessentially identical or essentially complementary to a Polyphenoloxidase (PPO) gene or transcript of the Polyphenol oxidase (PPO) genecan be generated by a variety of approaches, including but not limitedto, any of the tiling, least homology, or most homology approachesprovided herein. Such populations of polynucleotides can also begenerated or obtained from any of the polynucleotides or genes providedherewith provided in the sequence listing. Such populations ofpolynucleotides can also be generated or obtained from any genes thatare orthologous to the genes provided herewith in the sequence listing.Such polynucleotides can be topically applied to a surface of plants ina composition comprising at least one polynucleotide from saidpopulation and a transfer agent to obtain treated plants. Treated plantsthat exhibit suppression of the Polyphenol oxidase (PPO) gene and/orexhibit an improvement in improved shelf life and reduced postharvestlosses are identified, thus identifying a preferred polynucleotide thatimproves improved shelf life and reduced postharvest losses in a plant.Suppression of the Polyphenol oxidase (PPO) gene can be determined byany assay for the levels and/or activity of a Polyphenol oxidase (PPO)gene product (i.e., transcript or protein). Suitable assays fortranscripts include, but are not limited to, semi-quantitative orquantitative reverse transcriptase PCR® (qRT-PCR) assays. Suitableassays for proteins include, but are not limited to, semi-quantitativeor quantitative immunoassays, biochemical activity assays, or biologicalactivity assays. In certain embodiments, the polynucleotides can beapplied alone. In other embodiments, the polynucleotides can be appliedin pools of multiple polynucleotides. When a pool of polynucleotidesprovides for suppression of the Polyphenol oxidase (PPO) gene and/or animprovement in improved shelf life and reduced postharvest losses areidentified, the pool can be de-replicated and retested as necessary ordesired to identify one or more preferred polynucleotide(s) that improveimproved shelf life and reduced postharvest losses in a plant.

Methods of making polynucleotides are well known in the art. Suchmethods of making polynucleotides can include in vivo biosynthesis, invitro enzymatic synthesis, or chemical synthesis. In certainembodiments, RNA molecules can be made by either in vivo or in vitrosynthesis from DNA templates where a suitable promoter is operablylinked to the polynucleotide and a suitable DNA-dependent RNA polymeraseis provided. DNA-dependent RNA polymerases include, but are not limitedto, E. coli or other bacterial RNA polymerases as well as thebacteriophage RNA polymerases such as the T7, T3, and SP6 RNApolymerases. Commercial preparation of oligonucleotides often providestwo deoxyribonucleotides on the 3′ end of the sense strand. Longpolynucleotide molecules can be synthesized from commercially availablekits, for example, kits from Applied Biosystems/Ambion (Austin, Tex.)have DNA ligated on the 5′ end that encodes a bacteriophage T7polymerase promoter that makes RNA strands that can be assembled into adsRNA. Alternatively, dsRNA molecules can be produced from expressioncassettes in bacterial cells that have regulated or deficient RNase IIIenzyme activity. Long polynucleotide molecules can also be assembledfrom multiple RNA or DNA fragments. In some embodiments designparameters such as Reynolds score (Reynolds et al. Nature Biotechnology22, 326-330 (2004) and Tuschl rules (Pei and Tuschl, Nature Methods3(9): 670-676, 2006) are known in the art and are used in selectingpolynucleotide sequences effective in gene silencing. In someembodiments random design or empirical selection of polynucleotidesequences is used in selecting polynucleotide sequences effective ingene silencing. In some embodiments the sequence of a polynucleotide isscreened against the genomic DNA of the intended plant to minimizeunintentional silencing of other genes.

While there is no upper limit on the concentrations and dosages ofpolynucleotide molecules that can be useful in the methods andcompositions provided herein, lower effective concentrations and dosageswill generally be sought for efficiency. The concentrations can beadjusted in consideration of the volume of spray or treatment applied toplant leaves or other plant part surfaces, such as flower petals, stems,tubers, fruit, anthers, pollen, leaves, roots, or seeds. In oneembodiment, a useful treatment for herbaceous plants using 25-merpolynucleotide molecules is about 1 nanomole (nmol) of polynucleotidemolecules per plant, for example, from about 0.05 to 1 nmolpolynucleotides per plant. Other embodiments for herbaceous plantsinclude useful ranges of about 0.05 to about 100 nmol, or about 0.1 toabout 20 nmol, or about 1 nmol to about 10 nmol of polynucleotides perplant. In certain embodiments, about 40 to about 50 nmol of a ssDNApolynucleotide is applied. In certain embodiments, about 0.5 nmol toabout 2 nmol of a dsRNA is applied. In certain embodiments, acomposition containing about 0.5 to about 2.0 mg/mL, or about 0.14 mg/mLof dsRNA or ssDNA (21-mer) is applied. In certain embodiments, acomposition of about 0.5 to about 1.5 mg/mL of a long dsRNApolynucleotide (i.e., about 50 to about 200 or more nucleotides) isapplied. In certain embodiments, about 1 nmol to about 5nmol of a dsRNAis applied to a plant. In certain embodiments, the polynucleotidecomposition as topically applied to the plant contains the at least onepolynucleotide at a concentration of about 0.01 to about 10 milligramsper milliliter, or about 0.05 to about 2 milligrams per milliliter, orabout 0.1 to about 2 milligrams per milliliter. Very large plants,trees, or vines may require correspondingly larger amounts ofpolynucleotides. When using long dsRNA molecules that can be processedinto multiple oligonucleotides, lower concentrations can be used. Toillustrate embodiments of the invention, the factor 1×, when applied tooligonucleotide molecules is arbitrarily used to denote a treatment of0.8 nmol of polynucleotide molecule per plant; 10×, 8 nmol ofpolynucleotide molecule per plant; and 100×, 80 nmol of polynucleotidemolecule per plant.

The polynucleotide compositions of this invention are useful incompositions, such as liquids that comprise polynucleotide molecules,alone or in combination with other components either in the same liquidor in separately applied liquids that provide a transfer agent. As usedherein, a transfer agent is an agent that, when combined with apolynucleotide in a composition that is topically applied to a targetplant surface, enables the polynucleotide to enter a plant cell. Incertain embodiments, a transfer agent is an agent that conditions thesurface of plant tissue, e.g., seeds, leaves, stems, roots, flowers, orfruits, to permeation by the polynucleotide molecules into plant cells.The transfer of polynucleotides into plant cells can be facilitated bythe prior or contemporaneous application of apolynucleotide-transferring agent to the plant tissue. In someembodiments the transferring agent is applied subsequent to theapplication of the polynucleotide composition. The polynucleotidetransfer agent enables a pathway for polynucleotides through cuticle waxbarriers, stomata and/or cell wall or membrane barriers into plantcells. Suitable transfer agents to facilitate transfer of thepolynucleotide into a plant cell include agents that increasepermeability of the exterior of the plant or that increase permeabilityof plant cells to oligonucleotides or polynucleotides. Such agents tofacilitate transfer of the composition into a plant cell include achemical agent, or a physical agent, or combinations thereof. Chemicalagents for conditioning or transfer include (a) surfactants, (b) anorganic solvent or an aqueous solution or aqueous mixtures of organicsolvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils, (g)enzymes, or combinations thereof. Embodiments of the method canoptionally include an incubation step, a neutralization step (e.g., toneutralize an acid, base, or oxidizing agent, or to inactivate anenzyme), a rinsing step, or combinations thereof. Embodiments of agentsor treatments for conditioning of a plant to permeation bypolynucleotides include emulsions, reverse emulsions, liposomes, andother micellar-like compositions. Embodiments of agents or treatmentsfor conditioning of a plant to permeation by polynucleotides includecounter-ions or other molecules that are known to associate with nucleicacid molecules, e.g., inorganic ammonium ions, alkyl ammonium ions,lithium ions, polyamines such as spermine, spermidine, or putrescine,and other cations. Organic solvents useful in conditioning a plant topermeation by polynucleotides include DMSO, DMF, pyridine,N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane,polypropylene glycol, other solvents miscible with water or that willdissolve phosphonucleotides in non-aqueous systems (such as is used insynthetic reactions). Naturally derived or synthetic oils with orwithout surfactants or emulsifiers can be used, e.g., plant-sourcedoils, crop oils (such as those listed in the 9th Compendium of HerbicideAdjuvants, publicly available on the worldwide web (internet) atherbicide.adjuvants.com can be used, e.g., paraffinic oils, polyol fattyacid esters, or oils with short-chain molecules modified with amides orpolyamines such as polyethyleneimine or N-pyrrolidine. Transfer agentsinclude, but are not limited to, organosilicone preparations.

In certain embodiments, an organosilicone preparation that iscommercially available as Silwet® L-77 surfactant having CAS Number27306-78-1 and EPA Number: CAL.REG.NO. 5905-50073-AA, and currentlyavailable from Momentive Performance Materials, Albany, N.Y. can be usedto prepare a polynucleotide composition. In certain embodiments where aSilwet L-77 organosilicone preparation is used as a pre-spray treatmentof plant leaves or other plant surfaces, freshly made concentrations inthe range of about 0.015 to about 2 percent by weight (wt percent)(e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05,0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) are efficacious inpreparing a leaf or other plant surface for transfer of polynucleotidemolecules into plant cells from a topical application on the surface. Incertain embodiments of the methods and compositions provided herein, acomposition that comprises a polynucleotide molecule and anorganosilicone preparation comprising Silwet L-77 in the range of about0.015 to about 2 percent by weight (wt percent) (e.g., about 0.01,0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065,0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,2.1, 2.2, 2.3, 2.5 wt percent) is used or provided. In certainembodiments of the methods and compositions provided herein, acomposition that comprises a polynucleotide molecule and anorganosilicone preparation comprising Silwet L-77 in the range of about0.3 to about 1 percent by weight (wt percent) or about 0.5 to about 1%.by weight (wt percent) is used or provided.

In certain embodiments of the methods and compositions provided herein,a composition that comprises a polynucleotide molecule and anorganosilicone preparation comprising Silwet L-77 in the range of about0.3 to about 1 percent by weight (wt percent) or about 0.5 to about 1%.by weight (wt percent) is used or provided. In certain embodiments, anyof the commercially available organosilicone preparations provided inthe following Table 1 can be used as transfer agents in a polynucleotidecomposition. In certain embodiments where an organosilicone preparationof the Table is used as a pre-spray treatment of plant leaves or othersurfaces, freshly made concentrations in the range of about 0.015 toabout 2 percent by weight (wt percent) (e.g., about 0.01, 0.015, 0.02,0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075,0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.5 wt percent) are efficacious in preparing a leaf or other plantsurface for transfer of polynucleotide molecules into plant cells from atopical application on the surface. In certain embodiments of themethods and compositions provided herein, a composition that comprises apolynucleotide molecule and an organosilicone preparation of Table 1 inthe range of about 0.015 to about 2 percent by weight (wt percent)(e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05,0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 (wt percent) is used or provided.

TABLE 1 Exemplary organosilicone preparations Name CAS numberManufacturer ^(1,2) BREAK-THRU ® S 321 na Evonik Industries AGBREAK-THRU ® S 200  67674-67-3 Evonik Industries AG BREAK-THRU ® OE 441 68937-55-3 Evonik Industries AG BREAK-THRU ® S 278  27306-78-1 EvonikGoldschmidt BREAK-THRU ® S 243 na Evonik Industries AG Silwet ® L-77 27306-78-1 Momentive Performance Materials Silwet ® HS 429 na MomentivePerformance Materials Silwet ® HS 312 na Momentive Performance MaterialsBREAK-THRU ® S 233 134180-76-0 Evonik Industries AG Silwet ® HS 508Momentive Performance Materials Silwet ® HS 604 Momentive PerformanceMaterials ¹ Evonik Industries AG, Essen, Germany ² Momentive PerformanceMaterials, Albany, New York

Organosilicone preparations used in the methods and compositionsprovided herein can comprise one or more effective organosiliconecompounds. As used herein, the phrase “effective organosiliconecompound” is used to describe any organosilicone compound that is foundin an organosilicone preparation that enables a polynucleotide to entera plant cell. In certain embodiments, an effective organosiliconecompound can enable a polynucleotide to enter a plant cell in a mannerpermitting a polynucleotide mediated suppression of target geneexpression in the plant cell. In general, effective organosiliconecompounds include, but are not limited to, compounds that can comprise:i) a trisiloxane head group that is covalently linked to, ii) an alkyllinker including, but not limited to, an n-propyl linker, that iscovalently linked to, iii) a poly glycol chain, that is covalentlylinked to, iv) a terminal group. Trisiloxane head groups of sucheffective organosilicone compounds include, but are not limited to,heptamethyltrisiloxane. Alkyl linkers can include, but are not limitedto, an n-propyl linker. Poly glycol chains include, but are not limitedto, polyethylene glycol or polypropylene glycol. Poly glycol chains cancomprise a mixture that provides an average chain length “n” of about“7.5”. In certain embodiments, the average chain length “n” can varyfrom about 5 to about 14. Terminal groups can include, but are notlimited to, alkyl groups such as a methyl group. Effectiveorganosilicone compounds are believed to include, but are not limitedto, trisiloxane ethoxylate surfactants or polyalkylene oxide modifiedheptamethyl trisiloxane.

(Compound I: polyalkyleneoxide heptamethyltrisiloxane, average n=7.5).

One organosilicone compound believed to be ineffective comprises theformula:

In certain embodiments, an organosilicone preparation that comprises anorganosilicone compound comprising a trisiloxane head group is used inthe methods and compositions provided herein. In certain embodiments, anorganosilicone preparation that comprises an organosilicone compoundcomprising a heptamethyltrisiloxane head group is used in the methodsand compositions provided herein. In certain embodiments, anorganosilicone composition that comprises Compound I is used in themethods and compositions provided herein. In certain embodiments, anorganosilicone composition that comprises Compound I is used in themethods and compositions provided herein. In certain embodiments of themethods and compositions provided herein, a composition that comprises apolynucleotide molecule and one or more effective organosiliconecompound in the range of about 0.015 to about 2 percent by weight (wtpercent) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04,0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) is used orprovided.

In certain embodiments, the polynucleotide compositions that comprise anorganosilicone preparation can comprise a salt such as ammoniumchloride, tetrabutylphosphonium bromide, and/or ammonium sulfate.Ammonium chloride, tetrabutylphosphonium bromide, and/or ammoniumsulfate can be provided in the polynucleotide composition at aconcentration of about 0.5% to about 5% (w/v). An ammonium chloride,tetrabutylphosphonium bromide, and/or ammonium sulfate concentration ofabout 1% to about 3%, or about 2% (w/v) can also be used in thepolynucleotide compositions that comprise an organosilicone preparation.In certain embodiments, the polynucleotide compositions can comprise anammonium salt at a concentration greater or equal to 300 millimolar. Incertain embodiments, the polynucleotide compositions that comprise anorganosilicone preparation can comprise ammonium sulfate atconcentrations from about 80 to about 1200 mM or about 150 mM to about600 mM.

In certain embodiments, the polynucleotide compositions can alsocomprise a phosphate salt. Phosphate salts used in the compositionsinclude, but are not limited to, calcium, magnesium, potassium, orsodium phosphate salts. In certain embodiments, the polynucleotidecompositions can comprise a phosphate salt at a concentration of atleast about 5 millimolar, at least about 10 millimolar, or at leastabout 20 millimolar. In certain embodiments, the polynucleotidecompositions will comprise a phosphate salt in a range of about 1 mM toabout 25 mM or in a range of about 5 mM to about 25 mM. In certainembodiments, the polynucleotide compositions can comprise sodiumphosphate at a concentration of at least about 5 millimolar, at leastabout 10 millimolar, or at least about 20 millimolar. In certainembodiments, the polynucleotide compositions can comprise sodiumphosphate at a concentration of about 5 millimolar, about 10 millimolar,or about 20 millimolar. In certain embodiments, the polynucleotidecompositions will comprise a sodium phosphate salt in a range of about 1mM to about 25 mM or in a range of about 5 mM to about 25 mM. In certainembodiments, the polynucleotide compositions will comprise a sodiumphosphate salt in a range of about 10 mM to about 160 mM or in a rangeof about 20 mM to about 40 mM. In certain embodiments, thepolynucleotide compositions can comprise a sodium phosphate buffer at apH of about 6.8.

In certain embodiments, other useful transfer agents or adjuvants totransfer agents that can be used in polynucleotide compositions providedherein include surfactants and/or effective molecules contained therein.Surfactants and/or effective molecules contained therein include, butare not limited to, sodium or lithium salts of fatty acids (such astallow or tallowamines or phospholipids) and organosilicone surfactants.In certain embodiments, the polynucleotide compositions that comprise atransfer agent are formulated with counter-ions or other molecules thatare known to associate with nucleic acid molecules. Illustrativeexamples include tetraalkyl ammonium ions, trialkyl ammonium ions,sulfonium ions, lithium ions, and polyamines such as spermine,spermidine, or putrescine. In certain embodiments, the polynucleotidecompositions are formulated with a non-polynucleotide herbicide.Non-polynucleotide herbicidal molecules include, but are not limited to,glyphosate, auxin-like benzoic acid herbicides including dicamba,chloramben, and TBA, glufosinate, auxin-like herbicides includingphenoxy carboxylic acid herbicide, pyridine carboxylic acid herbicide,quinoline carboxylic acid herbicide, pyrimidine carboxylic acidherbicide, and benazolin-ethyl herbicide, sulfonylureas, imidazolinones,bromoxynil, delapon, cyclohezanedione, protoporphyrinogen oxidaseinhibitors, and 4-hydroxyphenyl-pyruvate-dioxygenase inhibitingherbicides.

In certain embodiments, the polynucleotides used in the compositionsthat are essentially identical or essentially complementary to thetarget gene or transcript will comprise the predominant nucleic acid inthe composition. Thus in certain embodiments, the polynucleotides thatare essentially identical or essentially complementary to the targetgene or transcript will comprise at least about 50%, 75%, 95%, 98%, or100% of the nucleic acids provided in the composition by either mass ormolar concentration. However, in certain embodiments, thepolynucleotides that are essentially identical or essentiallycomplementary to the target gene or transcript can comprise at leastabout 1% to about 50%, about 10% to about 50%, about 20% to about 50%,or about 30% to about 50% of the nucleic acids provided in thecomposition by either mass or molar concentration. Also provided arecompositions where the polynucleotides that are essentially identical oressentially complementary to the target gene or transcript can compriseat least about 1% to 100%, about 10% to 100%, about 20% to about 100%,about 30% to about 50%, or about 50% to 100% of the nucleic acidsprovided in the composition by either mass or molar concentration.

Polynucleotides comprising ssDNA, dsDNA, ssRNA, dsRNA, or RNA/DNAhybrids that are essentially identical or complementary to certain planttarget genes or transcripts and that can be used in compositionscontaining transfer agents that include, but are not limited to,organosilicone preparations, to suppress those target genes whentopically applied to plants are disclosed in co-assigned U.S. patentapplication Ser. No. 13/042856. Various polynucleotide herbicidalmolecules, compositions comprising those polynucleotide herbicidalmolecules and transfer agents that include, but are not limited to,organosilicone preparations, and methods whereby herbicidal effects areobtained by the topical application of such compositions to plants arealso disclosed in co-assigned U.S. patent application Ser. No.13/042856, and those polynucleotide herbicidal molecules, compositions,and methods are incorporated herein by reference in their entireties.Genes encoding proteins that can provide tolerance to an herbicideand/or that are targets of a herbicide are collectively referred toherein as “herbicide target genes”. Herbicide target genes include, butare not limited to, a 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS), a glyphosate oxidoreductase (GOX), a glyphosate decarboxylase,a glyphosate-N-acetyl transferase (GAT), a dicamba monooxygenase, aphosphinothricin acetyltransferase, a 2,2-dichloropropionic aciddehalogenase, an acetohydroxyacid synthase, an acetolactate synthase, ahaloarylnitrilase, an acetyl-coenzyme A carboxylase (ACCase), adihydropteroate synthase, a phytoene desaturase (PDS), a protoporphyrinIX oxygenase (PPO), a hydroxyphenylpyruvate dioxygenase (HPPD), apara-aminobenzoate synthase, a glutamine synthase, a cellulose synthase,a beta tubulin, and a serine hydroxymethyltransferase gene. The effectsof applying certain compositions comprising polynucleotides that areessentially identical or complementary to certain herbicide target genesand transfer agents on plants containing the herbicide target genes wasshown to be potentiated or enhanced by subsequent application of anherbicide that targets the same gene as the polynucleotide inco-assigned U.S. patent application Ser. No. 13/042856. For example,compositions comprising polynucleotides targeting the EPSPS herbicidetarget gene were potentiated by glyphosate in experiments disclosed inco-assigned U.S. patent application Ser. No. 13/042856.

In certain embodiments of the compositions and methods disclosed herein,the composition comprising a polynucleotide and a transfer agent canthus further comprise a second polynucleotide comprising at least 19contiguous nucleotides that are essentially identical or essentiallycomplementary to a transcript to a protein that confers resistance to aherbicide. In certain embodiments, the second polynucleotide does notcomprise a polynucleotide that is essentially identical or essentiallycomplementary to a transcript encoding a protein of a target plant thatconfers resistance to said herbicidal molecule. Thus, in an exemplaryand non-limiting embodiment, the second polynucleotide could beessentially identical or essentially complementary to a transcriptencoding a protein that confers resistance to a herbicide in a weed(such as an EPSPS encoding transcript) but would not be essentiallyidentical or essentially complementary to a transcript encoding aprotein that confers resistance to that same herbicide in a crop plant.

In certain embodiments, the polynucleotide compositions that comprise atransfer agent can comprise glycerin. Glycerin can be provided in thecomposition at a concentration of about 0.1% to about 1% (w/v or v/v). Aglycerin concentration of about 0.4% to about 0.6%, or about 0.5% (w/vor v/v) can also be used in the polynucleotide compositions thatcomprise a transfer agent.

In certain embodiments, the polynucleotide compositions that comprise atransfer agent can further comprise organic solvents. Such organicsolvents include, but are not limited to, DMSO, DMF, pyridine,N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane,polypropylene glycol, other solvents miscible with water or that willdissolve phosphonucleotides in non-aqueous systems (such as is used insynthetic reactions).

In certain embodiments, the polynucleotide compositions that comprise atransfer agent can further comprise naturally derived or synthetic oilswith or without surfactants or emulsifiers. Such oils include, but arenot limited to, plant-sourced oils, crop oils (such as those listed inthe 9th Compendium of Herbicide Adjuvants, publicly available on line atwww.herbicide.adjuvants.com), paraffinic oils, polyol fatty acid esters,or oils with short-chain molecules modified with amides or polyaminessuch as polyethyleneimine or N-pyrrolidine.

In aspects of the invention, methods include one or more applications ofthe composition comprising a polynucleotide and a transfer agent or oneor more effective components contained therein. In certain embodimentsof the methods, one or more applications of a transfer agent or one ormore effective components contained therein can precede one or moreapplications of the composition comprising a polynucleotide and atransfer agent. In embodiments where a transfer agent and/or one or moreeffective molecules contained therein is used either by itself as apre-treatment or as part of a composition that includes apolynucleotide, embodiments of the polynucleotide molecules aredouble-stranded RNA oligonucleotides, single-stranded RNAoligonucleotides, double-stranded RNA polynucleotides, single-strandedRNA polynucleotides, double-stranded DNA oligonucleotides,single-stranded DNA oligonucleotides, double-stranded DNApolynucleotides, single-stranded DNA polynucleotides, chemicallymodified RNA or DNA oligonucleotides or polynucleotides or mixturesthereof.

Compositions and methods of the invention are useful for modulating orsuppressing the expression of an endogenous target gene or transgenictarget gene in a plant cell or plant. In certain embodiments of themethods and compositions provided herein, expression of Polyphenoloxidase (PPO) target genes can be suppressed completely, partiallyand/or transiently to result in improved shelf life and reducedpostharvest losses. In various embodiments, a target gene includescoding (protein-coding or translatable) sequence, non-coding(non-translatable) sequence, or both coding and non-coding sequence.Compositions of the invention can include polynucleotides andoligonucleotides designed to target multiple genes, or multiple segmentsof one or more genes. The target gene can include multiple consecutivesegments of a target gene, multiple non-consecutive segments of a targetgene, multiple alleles of a target gene, or multiple target genes fromone or more species. Examples of target genes of the present inventioninclude endogenous Polyphenol oxidase (PPO) genes and Polyphenol oxidase(PPO) transgenes.

Target Polyphenol oxidase (PPO) genes and plants containing those targetPolyphenol oxidase (PPO) genes can be obtained from: i) row crop plantsincluding, but are not limited to, corn, soybean, cotton, canola, sugarbeet, alfalfa, sugarcane, rice, and wheat; ii) vegetable plantsincluding, but not limited to, tomato, potato, sweet pepper, hot pepper,melon, watermelon, cucumber, eggplant, cauliflower, broccoli, lettuce,spinach, onion, peas, carrots, sweet corn, Chinese cabbage, leek,fennel, pumpkin, squash or gourd, radish, Brussels sprouts, tomatillo,garden beans, dry beans, or okra; iii) culinary plants including, butnot limited to, basil, parsley, coffee, or tea; iv) fruit plantsincluding but not limited to apple, pear, cherry, peach, plum, apricot,banana, plantain, table grape, wine grape, citrus, avocado, mango, orberry; v) a tree grown for ornamental or commercial use, including, butnot limited to, a fruit or nut tree; or, vi) an ornamental plant (e.g.,an ornamental flowering plant or shrub or turf grass). The methods andcompositions provided herein can also be applied to plants produced by acutting, cloning, or grafting process (i.e., a plant not grown from aseed) including fruit trees and plants that include, but are not limitedto, citrus, apples, avocados, tomatoes, eggplant, cucumber, melons,watermelons, and grapes as well as various ornamental plants. Such rowcrop, vegetable, culinary, fruit, tree, or ornamental plants exhibitingimprovements that result from suppressing expression of Polyphenoloxidase (PPO) gene are provided herein. Such row crop, vegetable,culinary, fruit, tree, or ornamental plant parts or processed plantproducts exhibiting improved shelf life and reduced postharvest lossesthat result from suppressing expression of Polyphenol oxidase (PPO) geneare also provided herein. Such plant parts can include, but are notlimited to, flowers, stems, tubers, fruit, anthers, meristems, ovules,pollen, leaves, or seeds. Such processed plant products obtained fromthe plant parts can include, but are not limited to, a meal, a pulp, afeed, or a food product.

An aspect of the invention provides a method for modulating expressionof a Polyphenol oxidase (PPO) gene in a plant including (a) conditioningof a plant to permeation by polynucleotides and (b) treatment of theplant with the polynucleotide molecules, wherein the polynucleotidemolecules include at least one segment of 18 or more contiguousnucleotides cloned from or otherwise identified from the targetPolyphenol oxidase (PPO) gene in either anti-sense or sense orientation,whereby the polynucleotide molecules permeate the interior of the plantand induce modulation of the target Polyphenol oxidase (PPO) gene. Theconditioning and polynucleotide application can be performed separatelyor in a single step. When the conditioning and polynucleotideapplication are performed in separate steps, the conditioning canprecede or can follow the polynucleotide application within minutes,hours, or days. In some embodiments more than one conditioning step ormore than one polynucleotide molecule application can be performed onthe same plant. In embodiments of the method, the segment can be clonedor identified from (a) coding (protein-encoding), (b) non-coding(promoter and other gene related molecules), or (c) both coding andnon-coding parts of the target Polyphenol oxidase (PPO) gene. Non-codingparts include DNA, such as promoter regions or the RNA transcribed bythe DNA that provide RNA regulatory molecules, including but not limitedto: introns, 5′ or 3′ untranslated regions, and microRNAs (miRNA),trans-acting siRNAs, natural anti-sense siRNAs, and other small RNAswith regulatory function or RNAs having structural or enzymatic functionincluding but not limited to: ribozymes, ribosomal RNAs, t-RNAs,aptamers, and riboswitches. In certain embodiments where thepolynucleotide used in the composition comprises a promoter sequenceessentially identical to, or essentially complementary to at least 18contiguous nucleotides of the promoter of the endogenous targetPolyphenol oxidase (PPO) gene, the promoter sequence of thepolynucleotide is not operably linked to another sequence that istranscribed from the promoter sequence.

Compositions comprising a polynucleotide and a transfer agent providedherein can be topically applied to a plant or plant part by anyconvenient method, e.g., spraying or coating with a powder, or with aliquid composition comprising any of an emulsion, suspension, orsolution. Such topically applied sprays or coatings can be of either allor of any a portion of the surface of the plant or plant part.Similarly, the compositions comprising a transfer agent or otherpre-treatment can in certain embodiments be applied to the plant orplant part by any convenient method, e.g., spraying or wiping asolution, emulsion, or suspension. Compositions comprising apolynucleotide and a transfer agent provided herein can be topicallyapplied to plant parts that include, but are not limited to, flowers,stems, tubers, meristems, ovules, fruit, anthers, pollen, leaves, roots,or seeds.

Application of compositions comprising a polynucleotide and a transferagent to seeds is specifically provided herein. Seeds can be contactedwith such compositions by spraying, misting, immersion, and the like.

In certain embodiments, application of compositions comprising apolynucleotide and a transfer agent to plants, plant parts, or seeds inparticular can provide for the improved shelf life and reducedpostharvest losses in progeny plants, plant parts, or seeds derived fromthose treated plants, plant parts, or seeds. In certain embodiments,progeny plants, plant parts, or seeds derived from those treated plants,plant parts, or seeds will exhibit improved shelf life and reducedpostharvest losses that result from suppressing expression of Polyphenoloxidase (PPO) gene. In certain embodiments, the methods and compositionsprovided herein can provide for improved shelf life and reducedpostharvest losses in progeny plants or seeds as a result ofepigenetically inherited suppression of Polyphenol oxidase (PPO) geneexpression. In certain embodiments, such progeny plants exhibit improvedshelf life and reduced postharvest losses from epigenetically inheritedsuppression of Polyphenol oxidase (PPO) gene expression that is notcaused by a transgene where the polynucleotide is operably linked to apromoter, a viral vector, or a copy of the polynucleotide that isintegrated into a non-native location in the chromosomal DNA of theplant. Without seeking to be limited by theory, progeny plants or seedsderived from those treated plants, plant parts, or seeds can exhibit animprovement in improved shelf life and reduced postharvest lossesthrough an epigenetic mechanism that provides for propagation of anepigenetic condition where suppression of Polyphenol oxidase (PPO) geneexpression occurs in the progeny plants, plant parts, or plant seeds. Incertain embodiments, progeny plants or seeds exhibiting improved shelflife and reduced postharvest losses as a result of epigeneticallyinherited suppression of Polyphenol oxidase (PPO) gene expression canalso exhibit increased methylation, and in particular, increasedmethylation of cytosine residues, in the endogenous Polyphenol oxidase(PPO) gene of the plant. Plant parts, including seeds, of the progenyplants that exhibit improved shelf life and reduced postharvest lossesas a result of epigenetically inherited suppression of Polyphenoloxidase (PPO) gene expression, can also in certain embodiments exhibitincreased methylation, and in particular, increased methylation ofcytosine residues, in the endogenous Polyphenol oxidase (PPO) gene. Incertain embodiments, DNA methylation levels in DNA encoding theendogenous Polyphenol oxidase (PPO) gene can be compared in plants thatexhibit the improved shelf life and reduced postharvest losses andcontrol plants that do not exhibit the improved shelf life and reducedpostharvest losses to correlate the presence of the improved shelf lifeand reduced postharvest losses to epigenetically inherited suppressionof Polyphenol oxidase (PPO) gene expression and to identify plants thatcomprise the epigenetically inherited improved shelf life and reducedpostharvest losses.

Various methods of spraying compositions on plants or plant parts can beused to topically apply to a plant surface a composition comprising apolynucleotide that comprises a transfer agent. In the field, acomposition can be applied with a boom that extends over the crops anddelivers the composition to the surface of the plants or with a boomlesssprayer that distributes a composition across a wide area. Agriculturalsprayers adapted for directional, broadcast, or banded spraying can alsobe used in certain embodiments. Sprayers adapted for spraying particularparts of plants including, but not limited to, leaves, the undersides ofleaves, flowers, stems, male reproductive organs such as tassels,meristems, pollen, ovules, and the like can also be used. Compositionscan also be delivered aerially, such as by a crop dusting airplane. Incertain embodiments, the spray can be delivered with a pressurizedbackpack sprayer calibrated to deliver the appropriate rate of thecomposition. In certain embodiments, such a backpack sprayer is a carbondioxide pressurized sprayer with a 11015 flat fan or equivalent spraynozzle with a customized single nozzle assembly (to minimize waste) at aspray pressure of about 0.25 MPa and/or any single nozzle sprayerproviding an effective spray swath of 60 cm above the canopy of 3 to 12inch tall growing plants can be used. Plants in a greenhouse or growthchamber can be treated using a track sprayer or laboratory sprayer witha 11001XR or equivalent spray nozzle to deliver the sample solution at adetermined rate. An exemplary and non-limiting rate is about 140 L/ha atabout 0.25 MPa pressure.

In certain embodiments, it is also contemplated that a plant part can besprayed with the composition comprising a polynucleotide that comprisesa transfer agent. Such plant parts can be sprayed either pre-orpost-harvest to provide improved shelf life and reduced postharvestlosses in the plant part that results from suppression of Polyphenoloxidase (PPO) gene expression. Compositions can be topically applied toplant parts attached to a plant by a spray as previously described.Compositions can be topically applied to plant parts that are detachedfrom a plant by a spray as previously described or by an alternativemethod. Alternative methods for applying compositions to detached partsinclude, but are not limited to, passing the plant parts through a sprayby a conveyor belt or trough, or immersing the plant parts in thecomposition.

Compositions comprising polynucleotides and transfer agents can beapplied to plants or plant parts at one or more developmental stages asdesired and/or as needed. Application of compositions to pre-germinationseeds and/or to post-germination seedlings is provided in certainembodiments. Seeds can be treated with polynucleotide compositionsprovided herein by methods including, but not limited to, spraying,immersion, or any process that provides for coating, imbibition, and/oruptake of the polynucleotide composition by the seed. Seeds can betreated with polynucleotide compositions using seed batch treatmentsystems or continuous flow treatment systems. Seed coating systems areat least described in U.S. Pat. Nos. 6,582,516, 5,891,246, 4,079,696,and 4,023,525. Seed treatment can also be effected in laboratory orcommercial scale treatment equipment such as a tumbler, a mixer, or apan granulator. A polynucleotide composition used to treat seeds cancontain one or more other desirable components including, but notlimited to liquid diluents, binders to serve as a matrix for thepolynucleotide, fillers for protecting the seeds during stressconditions, and plasticizers to improve flexibility, adhesion and/orspreadability of the coating. In addition, for oily polynucleotidecompositions containing little or no filler, drying agents such ascalcium carbonate, kaolin or bentonite clay, perlite, diatomaceous earthor any other adsorbent material can be added. Use of such components inseed treatments is described in U.S. Pat. No. 5,876,739. Additionalingredients can be incorporated into the polynucleotide compositionsused in seed treatments. Such ingredients include but are not limitedto: conventional sticking agents, dispersing agents such asmethylcellulose (Methocel A15LV or Methocel A15C, for example, serve ascombined dispersant/sticking agents for use in seed treatments),polyvinyl alcohol (e.g., Elvanol 51-05), lecithin (e.g., Yelkinol P),polymeric dispersants (e.g., polyvinylpyrrolidone/vinyl acetate PVPNAS-630), thickeners (e.g., clay thickeners such as Van Gel B to improveviscosity and reduce settling of particle suspensions), emulsionstabilizers, surfactants, antifreeze compounds (e.g., urea), dyes,colorants, and the like that can be combined with compositionscomprising a polynucleotide and a transfer agent. Further ingredientsused in compositions that can be applied to seeds can be found inMcCutcheon's, vol. 1, “Emulsifiers and Detergents,” MC PublishingCompany, Glen Rock, N.J., U.S.A., 1996 and in McCutcheon's, vol. 2,“Functional Materials,” MC Publishing Company, Glen Rock, N.J., U.S.A.,1996. Methods of applying compositions to seeds and pesticidalcompositions that can be used to treat seeds are described in U.S.Patent Application Publication No. 20080092256, which is incorporatedherein by reference in its entirety.

Application of the compositions in early, mid-, and late vegetativestages of plant development is provided in certain embodiments.Application of the compositions in early, mid, and late reproductivestages is also provided in certain embodiments. Application of thecompositions to plant parts at different stages of maturation is alsoprovided.

In certain embodiments, methods and polynucleotide compositions areprovided that can be applied to living plant cells/tissues to suppressexpression of a Polyphenol oxidase 11 (PPO11) gene and that provideimproved shelf life, reduced browning, reduced postharvest losses orcombinations thereof to a crop plant in need of the benefit. Alsoprovided herein are plants and plant parts exhibiting improved shelflife and reduced postharvest losses as well as processed products ofsuch plants or plant parts. In certain embodiments, the compositions maybe topically applied to the surface of a plant, such as to the surfaceof a leaf, tuber, or fruit, and the compositions may include a transferagent. Aspects of the method can be applied to various crops, forexample, including but not limited to lettuce, apples and potatoes. Themethods and compositions provided herein can also be applied to plantsproduced by a cutting, cloning, or grafting process (i.e., a plant notgrown from a seed) that include fruit trees and plants. Fruit treesproduced by such processes include, but are not limited to, citrus andapple trees. Plants produced by such processes include, but are notlimited to, avocados, tomatoes, eggplant, cucumber, melons, watermelons,and grapes as well as various ornamental plants.

Without being bound by theory, in certain embodiments, the compositionsand methods of the present invention are believed to operate through oneor more of the several natural cellular pathways involved inRNA-mediated gene suppression as generally described in Brodersen andVoinnet (2006), Trends Genetics, 22:268-280; Tomari and Zamore (2005)Genes & Dev., 19:517-529; Vaucheret (2006) Genes Dev., 20:759-771; Meinset al. (2005) Annu. Rev. Cell Dev. Biol., 21:297-318; and Jones-Rhoadeset al. (2006) Annu. Rev. Plant Biol., 57:19-53. RNA-mediated genesuppression generally involves a double-stranded RNA (dsRNA)intermediate that is formed intra-molecularly within a single RNAmolecule or inter-molecularly between two RNA molecules. This longerdsRNA intermediate is processed by a ribonuclease of the RNAase IIIfamily (Dicer or Dicer-like ribonuclease) to one or more shorterdouble-stranded RNAs, one strand of which is incorporated into theRNA-induced silencing complex (“RISC”). For example, the siRNA pathwayinvolves the cleavage of a longer double-stranded RNA intermediate tosmall interfering RNAs (“siRNAs”). The size of siRNAs is believed torange from about 19 to about 25 base pairs, but the most common classesof siRNAs in plants include those containing 21 to 24 base pairs (See,Hamilton et al. (2002) EMBO J., 21:4671-4679).

Plant Transformation Constructs

Vectors used for plant transformation may include, for example,plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterialartificial chromosomes) or any other suitable cloning system, as well asfragments of DNA therefrom. Thus when the term “vector” or “expressionvector” is used, all of the foregoing types of vectors, as well asnucleic acid sequences isolated therefrom, are included. It iscontemplated that utilization of cloning systems with large insertcapacities will allow introduction of large DNA sequences comprisingmore than one selected gene. In accordance with the invention, thiscould be used to introduce genes corresponding to an entire biosyntheticpathway into a plant. Introduction of such sequences may be facilitatedby use of bacterial or yeast artificial chromosomes (BACs or YACs,respectively), or even plant artificial chromosomes. For example, theuse of BACs for Agrobacterium-mediated transformation was disclosed byHamilton et al., Proc. Natl. Acad. Sci. USA, 93(18):9975-9979, 1996.

Particularly useful for transformation are expression cassettes whichhave been isolated from such vectors. DNA segments used for transformingplant cells will, of course, generally comprise the cDNA, gene or geneswhich one desires to introduce into and have expressed in the hostcells. These DNA segments can further include structures such aspromoters, enhancers, polylinkers, or even regulatory genes as desired.The DNA segment or gene chosen for cellular introduction will oftenencode a protein which will be expressed in the resultant recombinantcells resulting in a screenable or selectable trait and/or which willimpart an improved phenotype to the resulting transgenic plant. However,this may not always be the case, and the present invention alsoencompasses transgenic plants incorporating non-expressed transgenes.Preferred components likely to be included with vectors used in thecurrent invention are as follows.

A. Regulatory Elements

Exemplary promoters for expression of a nucleic acid sequence includeplant promoter such as the CaMV 35S promoter (Odell et al., 1985), orothers such as CaMV 19S (Lawton et al., 1987), nos (Ebert et al., 1987),Adh (Walker et al., 1987), sucrose synthase (Yang and Russell, 1990), atubulin, actin (Wang et al., 1992), cab (Sullivan et al., 1989), PEPCase(Hudspeth and Grula, 1989) or those associated with the R gene complex(Chandler et al., 1989). Tissue specific promoters such as root cellpromoters (Conkling et al., 1990) and tissue specific enhancers (Frommet al., 1986) are also contemplated to be useful, as are induciblepromoters such as ABA- and turgor-inducible promoters. The PAL2 promotermay in particular be useful with the invention (U.S. Pat. Appl. Pub.2004/0049802, the entire disclosure of which is specificallyincorporated herein by reference).

The DNA sequence between the transcription initiation site and the startof the coding sequence, i.e., the untranslated leader sequence, can alsoinfluence gene expression. One may thus wish to employ a particularleader sequence with a transformation construct of the invention.Preferred leader sequences are contemplated to include those whichcomprise sequences predicted to direct optimum expression of theattached gene, i.e., to include a preferred consensus leader sequencewhich may increase or maintain mRNA stability and prevent inappropriateinitiation of translation. The choice of such sequences will be known tothose of skill in the art in light of the present disclosure. Sequencesthat are derived from genes that are highly expressed in plants willtypically be preferred.

It is contemplated that vectors for use in accordance with the presentinvention may be constructed to include an ocs enhancer element. Thiselement was first identified as a 16 bp palindromic enhancer from theoctopine synthase (ocs) gene of Agrobacterium (Ellis et al., 1987), andis present in at least 10 other promoters (Bouchez et al., 1989). Theuse of an enhancer element, such as the ocs element and particularlymultiple copies of the element, may act to increase the level oftranscription from adjacent promoters when applied in the context ofplant transformation.

It is envisioned that polynucleotide sequences or selected DNA sequencesmay be introduced under the control of novel promoters or enhancers,etc., or homologous or tissue specific promoters or control elements.Vectors for use in tissue-specific targeting of genes in transgenicplants will typically include tissue-specific promoters and may alsoinclude other tissue-specific control elements such as enhancersequences. Promoters which direct specific or enhanced expression incertain plant tissues will be known to those of skill in the art inlight of the present disclosure. These include, for example, the rbcSpromoter, specific for green tissue; the ocs, nos and mas promoterswhich have higher activity in roots or wounded leaf tissue. In certainembodiments, promoters can be employed that cause low expression of theselected DNA. Low expression promoters can be obtained by mutationand/or recombination of DNA elements of promoters that cause highexpression or by selecting upstream regulatory elements of genes thatcause expression of mRNA or protein with low abundance.

B. Terminators

Transformation constructs prepared in accordance with the invention willtypically include a 3′ end DNA sequence that acts as a signal toterminate transcription and allow for the poly-adenylation of the mRNAproduced by coding sequences operably linked to a promoter. Examples ofterminators that are deemed to be useful in this context include thosefrom the nopaline synthase gene of Agrobacterium tumefaciens (nos 3′end) (Bevan et al., 1983), the terminator for the T7 transcript from theoctopine synthase gene of Agrobacterium tumefaciens, and the 3′ end ofthe protease inhibitor I or II genes from potato or tomato. Regulatoryelements such as an Adh intron (Callis et al., 1987), sucrose synthaseintron (Vasil et al., 1989) or TMV omega element (Gallie et al., 1989),may further be included where desired.

C. Transit or Signal Peptides

Sequences that are joined to the coding sequence of an expressed gene,which are removed post-translationally from the initial translationproduct and which facilitate the transport of the protein into orthrough intracellular or extracellular membranes, are termed transit(usually into vacuoles, vesicles, plastids and other intracellularorganelles) and signal sequences (usually to the endoplasmic reticulum,golgi apparatus and outside of the cellular membrane). By facilitatingthe transport of the protein into compartments inside and outside thecell, these sequences may increase the accumulation of gene productprotecting them from proteolytic degradation. These sequences also allowfor additional mRNA sequences from highly expressed genes to be attachedto the coding sequence of the genes. Since mRNA being translated byribosomes is more stable than naked mRNA, the presence of translatablemRNA in front of the gene may increase the overall stability of the mRNAtranscript from the gene and thereby increase synthesis of the geneproduct. Since transit and signal sequences are usuallypost-translationally removed from the initial translation product, theuse of these sequences allows for the addition of extra translatedsequences that may not appear on the final polypeptide. It further iscontemplated that targeting of certain proteins may be desirable inorder to enhance the stability of the protein (U.S. Pat. No. 5,545,818,incorporated herein by reference in its entirety).

Additionally, vectors may be constructed and employed in theintracellular targeting of a specific gene product within the cells of atransgenic plant or in directing a protein to the extracellularenvironment. This generally will be achieved by joining a DNA sequenceencoding a transit or signal peptide sequence to the coding sequence ofa particular gene. The resultant transit, or signal, peptide willtransport the protein to a particular intracellular, or extracellulardestination, respectively, and will then be post-translationallyremoved.

D. Marker Genes

By employing a selectable or screenable marker protein, one can provideor enhance the ability to identify transformants. “Marker genes” aregenes that impart a distinct phenotype to cells expressing the markerprotein and thus allow such transformed cells to be distinguished fromcells that do not have the marker. Such genes may encode either aselectable or screenable marker, depending on whether the marker confersa trait which one can “select” for by chemical means, i.e., through theuse of a selective agent (e.g., a herbicide, antibiotic, or the like),or whether it is simply a trait that one can identify throughobservation or testing, i.e., by “screening” (e.g., the greenfluorescent protein). Of course, many examples of suitable markerproteins are known to the art and can be employed in the practice of theinvention.

Included within the terms selectable or screenable markers also aregenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers which are secretable antigens that can be identified byantibody interaction, or even secretable enzymes which can be detectedby their catalytic activity. Secretable proteins fall into a number ofclasses, including small, diffusible proteins detectable, e.g., byELISA; small active enzymes detectable in extracellular solution (e.g.,alpha-amylase, beta-lactamase, phosphinothricin acetyltransferase); andproteins that are inserted or trapped in the cell wall (e.g., proteinsthat include a leader sequence such as that found in the expression unitof extensin or tobacco PR S).

Many selectable marker coding regions are known and could be used withthe present invention including, but not limited to, neo (Potrykus etal., 1985), which provides kanamycin resistance and can be selected forusing kanamycin, G418, paromomycin, etc.; bar, which confers bialaphosor phosphinothricin resistance; a mutant EPSP synthase protein (Hincheeet al., 1988) conferring glyphosate resistance; a nitrilase such as bxnfrom Klebsiella ozaenae which confers resistance to bromoxynil (Stalkeret al., 1988); a mutant acetolactate synthase (ALS) which confersresistance to imidazolinone, sulfonylurea or other ALS inhibitingchemicals (European Patent Application 154, 204, 1985); a methotrexateresistant DHFR (Thillet et al., 1988), a dalapon dehalogenase thatconfers resistance to the herbicide dalapon; or a mutated anthranilatesynthase that confers resistance to 5-methyl tryptophan.

An illustrative embodiment of selectable marker capable of being used insystems to select transformants are those that encode the enzymephosphinothricin acetyltransferase, such as the bar gene fromStreptomyces hygroscopicus or the pat gene from Streptomycesviridochromogenes. The enzyme phosphinothricin acetyl transferase (PAT)inactivates the active ingredient in the herbicide bialaphos,phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami etal., 1986; Twell et al., 1989) causing rapid accumulation of ammonia andcell death.

Screenable markers that may be employed include a beta-glucuronidase(GUS) or uidA gene which encodes an enzyme for which various chromogenicsubstrates are known; an R-locus gene, which encodes a product thatregulates the production of anthocyanin pigments (red color) in planttissues (Dellaporta et al., 1988); a beta-lactamase gene (Sutcliffe,1978), which encodes an enzyme for which various chromogenic substratesare known (e.g., PADAC, a chromogenic cephalosporin); a xylE gene(Zukowsky et al., 1983) which encodes a catechol dioxygenase that canconvert chromogenic catechols; an alpha-amylase gene (Ikuta et al.,1990); a tyrosinase gene (Katz et al., 1983) which encodes an enzymecapable of oxidizing tyrosine to DOPA and dopaquinone which in turncondenses to form the easily-detectable compound melanin; abeta-galactosidase gene, which encodes an enzyme for which there arechromogenic substrates; a luciferase (lux) gene (Ow et al., 1986), whichallows for bioluminescence detection; an aequorin gene (Prasher et al.,1985) which may be employed in calcium-sensitive bioluminescencedetection; or a gene encoding for green fluorescent protein (Sheen etal., 1995; Haseloff et al., 1997; Reichel et al., 1996; Tian et al.,1997; WO 97/41228). The gene that encodes green fluorescent protein(GFP) is also contemplated as a particularly useful reporter gene (Sheenet al., 1995; Haseloff et al., 1997; Reichel et al., 1996; Tian et al.,1997; WO 97/41228). Expression of green fluorescent protein may bevisualized in a cell or plant as fluorescence following illumination byparticular wavelengths of light.

Antisense and RNAi Constructs

Antisense and RNAi treatments represent one way of altering PPO11activity in accordance with the invention. In particular, constructscomprising a PPO11 biosynthesis coding sequence, including fragmentsthereof, in antisense orientation, or combinations of sense andantisense orientation, may be used to decrease or effectively eliminatethe expression of a PPO11 gene in a plant and obtain an improvement inshelf life as is described herein. Accordingly, this may be used to“knock-out” the PPO11 or homologous sequences thereof.

Techniques for RNAi are well known in the art and are described in, forexample, Lehner et al., (2004) and Downward (2004). The technique isbased on the fact that double stranded RNA is capable of directing thedegradation of messenger RNA with sequence complementary to one or theother strand (Fire et al., 1998). Therefore, by expression of aparticular coding sequence in sense and antisense orientation, either asa fragment or longer portion of the corresponding coding sequence, theexpression of that coding sequence can be down-regulated.

Antisense, and in some aspects RNAi, methodology takes advantage of thefact that nucleic acids tend to pair with “complementary” sequences. Bycomplementary, it is meant that polynucleotides are those which arecapable of base-pairing according to the standard Watson-Crickcomplementarity rules. That is, the larger purines will base pair withthe smaller pyrimidines to form combinations of guanine paired withcytosine (G:C) and adenine paired with either thymine (A:T) in the caseof DNA, or adenine paired with uracil (A:U) in the case of RNA.Inclusion of less common bases such as inosine, 5-methylcytosine,6-methyladenine, hypoxanthine and others in hybridizing sequences doesnot interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense oligonucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense and RNAi constructs, or DNA encoding such RNA's,may be employed to inhibit gene transcription or translation or bothwithin a host cell, either in vitro or in vivo, such as within a hostplant cell. In certain embodiments of the invention, such anoligonucleotide may comprise any unique portion of a nucleic acidsequence provided herein. In certain embodiments of the invention, sucha sequence comprises at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 or more contiguous nucleic acids of the nucleic acidsequence of a PPO11 gene, and/or complements thereof, which may be insense and/or antisense orientation. By including sequences in both senseand antisense orientation, increased suppression of the correspondingcoding sequence may be achieved.

Constructs may be designed that are complementary to all or part of thepromoter and other control regions, exons, introns or even exon-intronboundaries of a gene. It is contemplated that the most effectiveconstructs will include regions complementary to intron/exon splicejunctions. Thus, it is proposed that a preferred embodiment includes aconstruct with complementarity to regions within 50-200 bases of anintron-exon splice junction. It has been observed that some exonsequences can be included in the construct without seriously affectingthe target selectivity thereof. The amount of exonic material includedwill vary depending on the particular exon and intron sequences used.One can readily test whether too much exon DNA is included simply bytesting the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

As stated above, “complementary” or “antisense” means polynucleotidesequences that are substantially complementary over their entire lengthand have very few base mismatches. For example, sequences of fifteenbases in length may be termed complementary when they have complementarynucleotides at thirteen or fourteen positions. Naturally, sequenceswhich are completely complementary will be sequences which are entirelycomplementary throughout their entire length and have no basemismatches. Other sequences with lower degrees of homology also arecontemplated. For example, an RNAi or antisense construct which haslimited regions of high homology, but also contains a non-homologousregion (e.g., ribozyme; see above) could be designed. These molecules,though having less than 50% homology, would bind to target sequencesunder appropriate conditions.

It may be advantageous to combine portions of genomic DNA with cDNA orsynthetic sequences to generate specific constructs. For example, wherean intron is desired in the ultimate construct, a genomic clone willneed to be used. The cDNA or a synthesized polynucleotide may providemore convenient restriction sites for the remaining portion of theconstruct and, therefore, would be used for the rest of the sequence.

Methods for Genetic Transformation

Suitable methods for transformation of plant or other cells for use withthe current invention are believed to include virtually any method bywhich DNA can be introduced into a cell, such as by direct delivery ofDNA such as by PEG-mediated transformation of protoplasts (Omirulleh etal., 1993), by desiccation/inhibition-mediated DNA uptake (Potrykus etal., 1985), by electroporation (U.S. Pat. No. 5,384,253, specificallyincorporated herein by reference in its entirety), by agitation withsilicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. No. 5,302,523,specifically incorporated herein by reference in its entirety; and U.S.Pat. No. 5,464,765, specifically incorporated herein by reference in itsentirety), by Agrobacterium-mediated transformation (U.S. Pat. No.5,591,616 and U.S. Pat. No. 5,563,055; both specifically incorporatedherein by reference) and by acceleration of DNA coated particles (U.S.Pat. No. 5,550,318; U.S. Pat. No. 5,538,877; and U.S. Pat. No.5,538,880; each specifically incorporated herein by reference in itsentirety), etc. Through the application of techniques such as these, thecells of virtually any plant species may be stably transformed, andthese cells developed into transgenic plants.

A. Agrobacterium-Mediated Transformation

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described by Fraley etal., (1985), Rogers et al., (1987) and U.S. Pat. No. 5,563,055,specifically incorporated herein by reference in its entirety.

Agrobacterium-mediated transformation is most efficient indicotyledonous plants and is the preferable method for transformation ofdicots, including Arabidopsis, tobacco, tomato, alfalfa and potato.Indeed, while Agrobacterium-mediated transformation has been routinelyused with dicotyledonous plants for a number of years, it has onlyrecently become applicable to monocotyledonous plants. Advances inAgrobacterium-mediated transformation techniques have now made thetechnique applicable to nearly all monocotyledonous plants. For example,Agrobacterium-mediated transformation techniques have now been appliedto rice (Hiei et al., 1997; U.S. Pat. No. 5,591,616, specificallyincorporated herein by reference in its entirety), wheat (McCormac etal., 1998), barley (Tingay et al., 1997; McCormac et al., 1998), alfalfa(Thomas et al., 1990) and maize (Ishidia et al., 1996).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described(Rogers et al., 1987) have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes. Inaddition, Agrobacterium containing both armed and disarmed Ti genes canbe used for the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

B. Electroporation

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wounding in acontrolled manner. Examples of some species which have been transformedby electroporation of intact cells include maize (U.S. Pat. No.5,384,253; Rhodes et al., 1995; D'Halluin et al., 1992), wheat (Zhou etal., 1993), tomato (Hou and Lin, 1996), soybean (Christou et al., 1987)and tobacco (Lee et al., 1989).

One also may employ protoplasts for electroporation transformation ofplants (Bates, 1994; Lazzeri, 1995). For example, the generation oftransgenic soybean plants by electroporation of cotyledon-derivedprotoplasts is described by Dhir and Widholm in Intl. Patent Appl. Publ.No. WO 9217598 (specifically incorporated herein by reference). Otherexamples of species for which protoplast transformation has beendescribed include barley (Lazerri, 1995), sorghum (Battraw et al.,1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) andtomato (Tsukada, 1989).

C. Microprojectile Bombardment

Another method for delivering transforming DNA segments to plant cellsin accordance with the invention is microprojectile bombardment (U.S.Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No. 5,610,042;and PCT Application WO 94/09699; each of which is specificallyincorporated herein by reference in its entirety). In this method,particles may be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, platinum, and preferably, gold. It is contemplated that insome instances DNA precipitation onto metal particles would not benecessary for DNA delivery to a recipient cell using microprojectilebombardment. However, it is contemplated that particles may contain DNArather than be coated with DNA. Hence, it is proposed that DNA-coatedparticles may increase the level of DNA delivery via particlebombardment but are not, in and of themselves, necessary.

For the bombardment, cells in suspension are concentrated on filters orsolid culture medium. Alternatively, immature embryos or other targetcells may be arranged on solid culture medium. The cells to be bombardedare positioned at an appropriate distance below the macroprojectilestopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System, whichcan be used to propel particles coated with DNA or cells through ascreen, such as a stainless steel or Nytex screen, onto a filter surfacecovered with monocot plant cells cultured in suspension. The screendisperses the particles so that they are not delivered to the recipientcells in large aggregates. Microprojectile bombardment techniques arewidely applicable, and may be used to transform virtually any plantspecies. Examples of species for which have been transformed bymicroprojectile bombardment include monocot species such as maize (PCTApplication WO 95/06128), barley (Ritala et al., 1994; Hensgens et al.,1993), wheat (U.S. Pat. No. 5,563,055, specifically incorporated hereinby reference in its entirety), rice (Hensgens et al., 1993), oat (Torbetet al., 1995; Torbet et al., 1998), rye (Hensgens et al., 1993),sugarcane (Bower et al., 1992), and sorghum (Casa et al., 1993; Hagio etal., 1991); as well as a number of dicots including tobacco (Tomes etal., 1990; Buising and Benbow, 1994), soybean (U.S. Pat. No. 5,322,783,specifically incorporated herein by reference in its entirety),sunflower (Knittel et al., 1994), peanut (Singsit et al., 1997), cotton(McCabe and Martine11, 1993), tomato (VanEck et al., 1995), and legumesin general (U.S. Pat. No. 5,563,055, specifically incorporated herein byreference in its entirety).

D. Other Transformation Methods

Transformation of protoplasts can be achieved using methods based oncalcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Omirulleh et al., 1993; Frommet al., 1986; Uchimiya et al., 1986; Callis et al., 1987; Marcotte etal., 1988).

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of cereals from protoplastshave been described (Toriyama et al., 1986; Yamada et al., 1986;Abdullah et al., 1986; Omirulleh et al., 1993 and U.S. Pat. No.5,508,184; each specifically incorporated herein by reference in itsentirety). Examples of the use of direct uptake transformation of cerealprotoplasts include transformation of rice (Ghosh-Biswas et al., 1994),sorghum (Battraw and Hall, 1991), barley (Lazerri, 1995), oat (Zheng andEdwards, 1990) and maize (Omirulleh et al., 1993).

To transform plant strains that cannot be successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuescan be utilized. For example, regeneration of cereals from immatureembryos or explants can be effected as described (Vasil, 1989). Also,silicon carbide fiber-mediated transformation may be used with orwithout protoplasting (Kaeppler, 1990; Kaeppler et al., 1992; U.S. Pat.No. 5,563,055, specifically incorporated herein by reference in itsentirety). Transformation with this technique is accomplished byagitating silicon carbide fibers together with cells in a DNA solution.DNA passively enters as the cells are punctured. This technique has beenused successfully with, for example, the monocot cereals maize (PCTApplication WO 95/06128, specifically incorporated herein by referencein its entirety; (Thompson, 1995) and rice (Nagatani, 1997).

E. Tissue Cultures

Tissue cultures may be used in certain transformation techniques for thepreparation of cells for transformation and for the regeneration ofplants therefrom. Maintenance of tissue cultures requires use of mediaand controlled environments. “Media” refers to the numerous nutrientmixtures that are used to grow cells in vitro, that is, outside of theintact living organism. The medium usually is a suspension of variouscategories of ingredients (salts, amino acids, growth regulators,sugars, buffers) that are required for growth of most cell types.However, each specific cell type requires a specific range of ingredientproportions for growth, and an even more specific range of formulas foroptimum growth. Rate of cell growth also will vary among culturesinitiated with the array of media that permit growth of that cell type.

Nutrient media is prepared as a liquid, but this may be solidified byadding the liquid to materials capable of providing a solid support.Agar is most commonly used for this purpose. Bactoagar, Hazelton agar,Gelrite, and Gelgro are specific types of solid support that aresuitable for growth of plant cells in tissue culture.

Some cell types will grow and divide either in liquid suspension or onsolid media. As disclosed herein, plant cells will grow in suspension oron solid medium, but regeneration of plants from suspension culturestypically requires transfer from liquid to solid media at some point indevelopment. The type and extent of differentiation of cells in culturewill be affected not only by the type of media used and by theenvironment, for example, pH, but also by whether media is solid orliquid.

Tissue that can be grown in a culture includes meristem cells, Type I,Type II, and Type III callus, immature embryos and gametic cells such asmicrospores, pollen, sperm and egg cells. Type I, Type II, and Type IIIcallus may be initiated from tissue sources including, but not limitedto, immature embryos, seedling apical meristems, root, leaf, microsporesand the like. Those cells which are capable of proliferating as callusalso are recipient cells for genetic transformation.

Somatic cells are of various types. Embryogenic cells are one example ofsomatic cells which may be induced to regenerate a plant through embryoformation. Non-embryogenic cells are those which typically will notrespond in such a fashion. Certain techniques may be used that enrichrecipient cells within a cell population. For example, Type II callusdevelopment, followed by manual selection and culture of friable,embryogenic tissue, generally results in an enrichment of cells. Manualselection techniques which can be employed to select target cells mayinclude, e.g., assessing cell morphology and differentiation, or may usevarious physical or biological means. Cryopreservation also is apossible method of selecting for recipient cells.

Manual selection of recipient cells, e.g., by selecting embryogeniccells from the surface of a Type II callus, is one means that may beused in an attempt to enrich for particular cells prior to culturing(whether cultured on solid media or in suspension).

Where employed, cultured cells may be grown either on solid supports orin the form of liquid suspensions. In either instance, nutrients may beprovided to the cells in the form of media, and environmental conditionscontrolled. There are many types of tissue culture media comprised ofvarious amino acids, salts, sugars, growth regulators and vitamins. Mostof the media employed in the practice of the invention will have somesimilar components, but may differ in the composition and proportions oftheir ingredients depending on the particular application envisioned.For example, various cell types usually grow in more than one type ofmedia, but will exhibit different growth rates and differentmorphologies, depending on the growth media. In some media, cellssurvive but do not divide. Various types of media suitable for cultureof plant cells previously have been described. Examples of these mediainclude, but are not limited to, the N6 medium described by Chu et al.(1975) and MS media (Murashige and Skoog, 1962).

Production and Characterization of Stably Transformed Plants

After effecting delivery of exogenous DNA to recipient cells, the nextsteps generally concern identifying the transformed cells for furtherculturing and plant regeneration. In order to improve the ability toidentify transformants, one may desire to employ a selectable orscreenable marker gene with a transformation vector prepared inaccordance with the invention. In this case, one would then generallyassay the potentially transformed cell population by exposing the cellsto a selective agent or agents, or one would screen the cells for thedesired marker gene trait.

A. Selection

It is believed that DNA is introduced into only a small percentage oftarget cells in any one study. In order to provide an efficient systemfor identification of those cells receiving DNA and integrating it intotheir genomes one may employ a means for selecting those cells that arestably transformed. One exemplary embodiment of such a method is tointroduce into the host cell, a marker gene which confers resistance tosome normally inhibitory agent, such as an antibiotic or herbicide.Examples of antibiotics which may be used include the aminoglycosideantibiotics neomycin, kanamycin and paromomycin, or the antibiotichygromycin. Resistance to the aminoglycoside antibiotics is conferred byaminoglycoside phosphostransferase enzymes such as neomycinphosphotransferase II (NPT II) or NPT I, whereas resistance tohygromycin is conferred by hygromycin phosphotransferase.

Potentially transformed cells then are exposed to the selective agent.In the population of surviving cells will be those cells where,generally, the resistance-conferring gene has been integrated andexpressed at sufficient levels to permit cell survival. Cells may betested further to confirm stable integration of the exogenous DNA.

One herbicide which constitutes a desirable selection agent is the broadspectrum herbicide bialaphos. Bialaphos is a tripeptide antibioticproduced by Streptomyces hygroscopicus and is composed ofphosphinothricin (PPT), an analogue of L-glutamic acid, and twoL-alanine residues. Upon removal of the L-alanine residues byintracellular peptidases, the PPT is released and is a potent inhibitorof glutamine synthetase (GS), a pivotal enzyme involved in ammoniaassimilation and nitrogen metabolism (Ogawa et al., 1973). SyntheticPPT, the active ingredient in the herbicide Liberty also is effective asa selection agent. Inhibition of GS in plants by PPT causes the rapidaccumulation of ammonia and death of the plant cells.

The organism producing bialaphos and other species of the genusStreptomyces also synthesizes an enzyme phosphinothricin acetyltransferase (PAT) which is encoded by the bar gene in Streptomyceshygroscopicus and the pat gene in Streptomyces viridochromogenes. Theuse of the herbicide resistance gene encoding phosphinothricin acetyltransferase (PAT) is referred to in DE 3642 829 A, wherein the gene isisolated from Streptomyces viridochromogenes. In the bacterial sourceorganism, this enzyme acetylates the free amino group of PPT preventingauto-toxicity (Thompson et al., 1987). The bar gene has been cloned(Murakami et al., 1986; Thompson et al., 1987) and expressed intransgenic tobacco, tomato, potato (De Block et al., 1987) Brassica (DeBlock et al., 1989) and maize (U.S. Pat. No. 5,550,318). In previousreports, some transgenic plants which expressed the resistance gene werecompletely resistant to commercial formulations of PPT and bialaphos ingreenhouses.

Another example of a herbicide which is useful for selection oftransformed cell lines in the practice of the invention is the broadspectrum herbicide glyphosate. Glyphosate inhibits the action of theenzyme EPSPS which is active in the aromatic amino acid biosyntheticpathway. Inhibition of this enzyme leads to starvation for the aminoacids phenylalanine, tyrosine, and tryptophan and secondary metabolitesderived thereof. U.S. Pat. No. 4,535,060 describes the isolation ofEPSPS mutations which confer glyphosate resistance on the Salmonellatyphimurium gene for EPSPS, aroA. The EPSPS gene was cloned from Zeamays and mutations similar to those found in a glyphosate resistant aroAgene were introduced in vitro. Mutant genes encoding glyphosateresistant EPSPS enzymes are described in, for example, InternationalPatent WO 97/4103. The best characterized mutant EPSPS gene conferringglyphosate resistance comprises amino acid changes at residues 102 and106, although it is anticipated that other mutations will also be useful(PCT/WO97/4103).

To use the bar-bialaphos or the EPSPS-glyphosate selective system,transformed tissue is cultured for 0-28 days on nonselective medium andsubsequently transferred to medium containing from 1-3 mg/l bialaphos or1-3 mM glyphosate as appropriate. While ranges of 1-3 mg/l bialaphos or1-3 mM glyphosate will typically be preferred, it is proposed thatranges of 0.1-50 mg/l bialaphos or 0.1-50 mM glyphosate will findutility.

An example of a screenable marker trait is the enzyme luciferase. In thepresence of the substrate luciferin, cells expressing luciferase emitlight which can be detected on photographic or x-ray film, in aluminometer (or liquid scintillation counter), by devices that enhancenight vision, or by a highly light sensitive video camera, such as aphoton counting camera. These assays are nondestructive and transformedcells may be cultured further following identification. The photoncounting camera is especially valuable as it allows one to identifyspecific cells or groups of cells which are expressing luciferase andmanipulate those in real time. Another screenable marker which may beused in a similar fashion is the gene coding for green fluorescentprotein.

B. Regeneration and Seed Production

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, may be cultured in mediathat supports regeneration of plants. In an exemplary embodiment, MS andN6 media may be modified by including further substances such as growthregulators. One such growth regulator is dicamba or 2,4-D. However,other growth regulators may be employed, including NAA, NAA+2,4-D orpicloram. Media improvement in these and like ways has been found tofacilitate the growth of cells at specific developmental stages. Tissuemay be maintained on a basic media with growth regulators untilsufficient tissue is available to begin plant regeneration efforts, orfollowing repeated rounds of manual selection, until the morphology ofthe tissue is suitable for regeneration, at least 2 wk, then transferredto media conducive to maturation of embryoids. Cultures are transferredevery 2 wk on this medium. Shoot development will signal the time totransfer to medium lacking growth regulators.

The transformed cells, identified by selection or screening and culturedin an appropriate medium that supports regeneration, will then beallowed to mature into plants. Developing plantlets are transferred tosoiless plant growth mix, and hardened, e.g., in an environmentallycontrolled chamber, for example, at about 85% relative humidity, 600 ppmCO2, and 25-250 microeinsteins m 2 s-1 of light. Plants may be maturedin a growth chamber or greenhouse. Plants can be regenerated from about6 wk to 10 months after a transformant is identified, depending on theinitial tissue. During regeneration, cells are grown on solid media intissue culture vessels. Illustrative embodiments of such vessels arepetri dishes and Plant Cons. Regenerating plants can be grown at about19 to 28° C. After the regenerating plants have reached the stage ofshoot and root development, they may be transferred to a greenhouse forfurther growth and testing.

Seeds on transformed plants may occasionally require embryo rescue dueto cessation of seed development and premature senescence of plants. Torescue developing embryos, they are excised from surface-disinfectedseeds 10-20 days post-pollination and cultured. An embodiment of mediaused for culture at this stage comprises MS salts, 2% sucrose, and 5.5g/l agarose. In embryo rescue, large embryos (defined as greater than 3mm in length) are germinated directly on an appropriate media. Embryossmaller than that may be cultured for 1 wk on media containing the aboveingredients along with 10-5M abscisic acid and then transferred togrowth regulator-free medium for germination.

C. Characterization

To confirm the presence of the exogenous DNA or “transgene(s)” in theregenerating plants, a variety of assays may be performed. Such assaysinclude, for example, “molecular biological” assays, such as Southernand Northern blotting and PCR™; “biochemical” assays, such as detectingthe presence of a protein product, e.g., by immunological means (ELISAsand Western blots) or by enzymatic function; plant part assays, such asleaf or root assays; and also, by analyzing the phenotype of the wholeregenerated plant.

D. DNA Integration, RNA Expression and Inheritance

Genomic DNA may be isolated from cell lines or any plant parts todetermine the presence of the exogenous gene through the use oftechniques well known to those skilled in the art. Note, that intactsequences will not always be present, presumably due to rearrangement ordeletion of sequences in the cell. The presence of DNA elementsintroduced through the methods of this invention may be determined, forexample, by polymerase chain reaction (PCR™). Using this technique,discreet fragments of DNA are amplified and detected by gelelectrophoresis. This type of analysis permits one to determine whethera gene is present in a stable transformant, but does not proveintegration of the introduced gene into the host cell genome. It istypically the case, however, that DNA has been integrated into thegenome of all transformants that demonstrate the presence of the genethrough PCR™ analysis. In addition, it is not typically possible usingPCR™ techniques to determine whether transformants have exogenous genesintroduced into different sites in the genome, i.e., whethertransformants are of independent origin. It is contemplated that usingPCR™ techniques it would be possible to clone fragments of the hostgenomic DNA adjacent to an introduced gene.

Positive proof of DNA integration into the host genome and theindependent identities of transformants may be determined using thetechnique of Southern hybridization. Using this technique specific DNAsequences that were introduced into the host genome and flanking hostDNA sequences can be identified. Hence the Southern hybridizationpattern of a given transformant serves as an identifying characteristicof that transformant. In addition it is possible through Southernhybridization to demonstrate the presence of introduced genes in highmolecular weight DNA, i.e., confirm that the introduced gene has beenintegrated into the host cell genome. The technique of Southernhybridization provides information that is obtained using PCR™, e.g.,the presence of a gene, but also demonstrates integration into thegenome and characterizes each individual transformant.

It is contemplated that using the techniques of dot or slot blothybridization which are modifications of Southern hybridizationtechniques one could obtain the same information that is derived fromPCR™, e.g., the presence of a gene.

Both PCR™ and Southern hybridization techniques can be used todemonstrate transmission of a transgene to progeny. In most instancesthe characteristic Southern hybridization pattern for a giventransformant will segregate in progeny as one or more Mendelian genes(Spencer et al., 1992) indicating stable inheritance of the transgene.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA will only be expressed in particular cells ortissue types and hence it will be necessary to prepare RNA for analysisfrom these tissues. PCR™techniques also may be used for detection andquantitation of RNA produced from introduced genes. In this applicationof PCR™ it is first necessary to reverse transcribe RNA into DNA, usingenzymes such as reverse transcriptase, and then through the use ofconventional PCR™ techniques amplify the DNA. In most instances PCR™techniques, while useful, will not demonstrate integrity of the RNAproduct. Further information about the nature of the RNA product may beobtained by Northern blotting. This technique will demonstrate thepresence of an RNA species and give information about the integrity ofthat RNA. The presence or absence of an RNA species also can bedetermined using dot or slot blot Northern hybridizations. Thesetechniques are modifications of Northern blotting and will onlydemonstrate the presence or absence of an RNA species.

E. Gene Expression

While Southern blotting and PCR™ may be used to detect the gene(s) inquestion, they do not provide information as to whether thecorresponding protein is being expressed. Expression may be evaluated byspecifically identifying the protein products of the introduced genes orevaluating the phenotypic changes brought about by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focusing, or by chromatographictechniques such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins offer opportunities for use ofspecific antibodies to detect their presence in formats such as an ELISAassay. Combinations of approaches may be employed with even greaterspecificity such as western blotting in which antibodies are used tolocate individual gene products that have been separated byelectrophoretic techniques. Additional techniques may be employed toabsolutely confirm the identity of the product of interest such asevaluation by amino acid sequencing following purification. Althoughthese are among the most commonly employed, other procedures may beadditionally used.

Assay procedures also may be used to identify the expression of proteinsby their functionality, especially the ability of enzymes to catalyzespecific chemical reactions involving specific substrates and products.These reactions may be followed by providing and quantifying the loss ofsubstrates or the generation of products of the reactions by physical orchemical procedures. Examples are as varied as the enzyme to be analyzedand may include assays for PAT enzymatic activity by followingproduction of radiolabeled acetylated phosphinothricin fromphosphinothricin and 14C-acetyl CoA or for anthranilate synthaseactivity by following loss of fluorescence of anthranilate, to name two.

Very frequently the expression of a gene product is determined byevaluating the phenotypic results of its expression. These assays alsomay take many forms including but not limited to analyzing changes inthe chemical composition, morphology, or physiological properties of theplant. Chemical composition may be altered by expression of genesencoding enzymes or storage proteins which change amino acid compositionand may be detected by amino acid analysis, or by enzymes which changestarch quantity which may be analyzed by near infrared reflectancespectrometry. Morphological changes may include greater stature orthicker stalks. Most often changes in response of plants or plant partsto imposed treatments are evaluated under carefully controlledconditions termed bioassays.

Breeding Plants of the Invention

In addition to direct transformation of a particular plant genotype witha construct prepared according to the current invention, transgenicplants may be made by crossing a plant having a selected DNA of theinvention to a second plant lacking the construct. For example, aselected lignin biosynthesis coding sequence can be introduced into aparticular plant variety by crossing, without the need for ever directlytransforming a plant of that given variety. Therefore, the currentinvention not only encompasses a plant directly transformed orregenerated from cells which have been transformed in accordance withthe current invention, but also the progeny of such plants.

As used herein the term “progeny” denotes the offspring of anygeneration of a parent plant prepared in accordance with the instantinvention, wherein the progeny comprises a selected DNA construct.“Crossing” a plant to provide a plant line having one or more addedtransgenes relative to a starting plant line, as disclosed herein, isdefined as the techniques that result in a transgene of the inventionbeing introduced into a plant line by crossing a starting line with adonor plant line that comprises a transgene of the invention. To achievethis one could, for example, perform the following steps:

-   -   (a) plant seeds of the first (starting line) and second (donor        plant line that comprises a transgene of the invention) parent        plants;    -   (b) grow the seeds of the first and second parent plants into        plants that bear flowers;    -   (c) pollinate a flower from the first parent plant with pollen        from the second parent plant; and    -   (d) harvest seeds produced on the parent plant bearing the        fertilized flower.

Backcrossing is herein defined as the process including the steps of:

-   -   (a) crossing a plant of a first genotype containing a desired        gene, DNA sequence or element to a plant of a second genotype        lacking the desired gene, DNA sequence or element;    -   (b) selecting one or more progeny plant containing the desired        gene, DNA sequence or element;    -   (c) crossing the progeny plant to a plant of the second        genotype; and    -   (d) repeating steps (b) and (c) for the purpose of transferring        a desired DNA sequence from a plant of a first genotype to a        plant of a second genotype.

Introgression of a DNA element into a plant genotype is defined as theresult of the process of backcross conversion. A plant genotype intowhich a DNA sequence has been introgressed may be referred to as abackcross converted genotype, line, inbred, or hybrid. Similarly a plantgenotype lacking the desired DNA sequence may be referred to as anunconverted genotype, line, inbred, or hybrid.

EXAMPLES Example 1 Total PPO Enzyme Activity Does Not Predict Shelf LifePerformance

A panel of 23 cultivars including discoloration controls were sown in athree replicate trial in a single environment. The cultivars wereharvested on the same day and total PPO enzymatic activity wasdetermined. A normal distribution of total units of PPO activity (U PPOActivity=uM*min-1*ug-1) was observed amongst the 23 cultivars. Thevarieties with the five highest and five lowest PPO activities were thengrown in a second environment, and shelf life was determined using a 6replicate randomized complete block design. It was recognized that theharvest activity did not predict shelf life performance, the two mostdiscoloration resistant varieties in this set were identified as havinghigh enzymatic activity. In a comparison of harvest PPO enzymaticactivity (total) for 8 different harvests of discoloration controls, 7of 8 comparisons were not statistically differentiated. These resultswere surprising and unexpected because Enzymatic activity, the sum totalof activity of all active enzymes, had previously been believed to be apredictor of shelf life.

Example 2 PPO11 Expression Increases over Time after Harvest

Eleven (11) PPO gene homologs, shown in FIGS. 1, and 3 PAL gene homologswere identified in lettuce. Gene expression analysis of thesediscoloration pathway enzyme homologs were evaluated for correlation todiscoloration phenotype/shelf life performance. The ability to correlatePPO enzyme activity at harvest or induction of specific pathway geneswith shelf life performance provided a focused selection tool to enablea breeding program to improve shelf life performance.

RT PCR protocol: QRT-PCRs were performed as described in the operator'smanual using a Stratagene MX3000PTM. Gene-specific PCR primers weredesigned using criteria including predicted melting temperature of atleast 58° C., primer length of 22-24 nucleotides, guanosine-cytosinecontent of at least 48%, and an amplicon length of 120-150 bp. Primerspecificity was confirmed by melting curve analysis, by an efficiency ofproduct amplification of 1.0§ 0.1, and by sequence verification of atleast eight cloned PCR amplicons for each gene. Reactions with waterinstead of cDNA template were run with each primer pair as control. Thestandard thermal profile: 95° C. for 10 min, then 60 cycles of 95° C.for 30 s, 53° C. for 30 s and 72° C. for 30 s was used. The fluorescencesignal was captured at the end of each cycle, and a melting curveanalysis was performed from the annealing temperature to 95° C. withdata capture every 0.2° C. during a 1 s hold. The quantity of eachtranscript is the average of two technical replicates. All amplificationplots were analyzed with the MX3000PTM software to obtain thresholdcycle (Ct) values. Transcript abundance was normalized to the transcriptabundance of ubiquitin (GenBank accession number EF681766).

The results are shown in FIG. 2, which provides a time-course experimentof the expression profile of PPOs. Two homologs of PPO, PPO2 and PPO011,were upregulated after harvest. PPO11 was the most upregulated PPOduring the time-course experiment. The expression of PPO1 and PPO10 wentdown over time. The expression of PPO8 did not change over time.

Example 3 Determining Shelf Life of Lettuce

The current industry standard for assessing lettuce performance,browning, or shelf life is to evaluate a lettuce variety in a modifiedatmosphere package at 14 days after processing and ensure that less than2% visible pinking or browning occurs. The performance of individualvarieties beyond 4 days has been unknown. The performance of choppedlettuce varieties in an ambient air assay at 4° C. is evaluated. Suchevaluation allows lettuce variety performance to be distinguished in arapid manner. In addition, correlation of polyphenol oxidase enzymatic(PPO) activity in the central rib of each variety with lettuceperformance was tested.

To carry out the shelf live evaluation, six heads of lettuce arecollected and packaged into a waxed cardboard box. A refrigerated truck,set to 41° F., is used for sample transport.

Individual heads are removed from the plastic bag and the ID tag placedinto a single blue tray. Two empty barcoded 50 ml tubes are placed intothe tray and the sample can then move on for chopping. Outer damagedleaves are removed and the base is cut off. The stem is removed anddiscarded, the inner heart leaves (yellow and less than 6 inches long)are discarded. From each head, two leaves from outer and inner leavesare set aside for central rib evaluation, 12 leaves in total arecollected. All of the remaining lettuce leaves are chopped for shelflife evaluation.

Central Rib evaluation: From the 12 petioles (6 heads×2 leaves) theupper leafy material and the material from the edge along the bottom ⅔of the lettuce rib are removed using a knife. The leafy material isdiscarded and the lower ⅔ of the central ribs are retained. All ribs arechopped into 1 inch slices, mixed by hand and then subsampled into tubesfilling to the 30 ml line.

Romaine, CRC, and spinach-type lettuce are prepared as follows. Theleafy material above the rib is removed. Excess leafy material parallelto the rib is removed to leave only about 1-2 inches of leafy materialon each side of the rib. If the leaf is narrow enough, this step is notperformed. The rib and flanking leafy pieces are chopped into ¾ inchpieces perpendicular to the rib length. All material is collected andimmersed into cold fresh water using a large screen basket, transferredinto an electric salad spinner and dried for 60 seconds. Three amplyfull quart clamshells (individual barcodes) are collected if available.Any remaining chopped lettuce is collected in a barcoded black tray.

Iceberg type lettuce: are to be prepared as follows: The lettuce ischopped into ¾ inch strips and then these strips are chopped at a 90°angle into ¾ inch squares. All material is collected into a blue bin,washed in mesh basket, and spun dry for 60 seconds in an electric saladspinner. Three amply full quart clamshells (individual barcodes) arecollected if available. Any remaining chopped lettuce is collected in abarcoded black tray. The samples are scanned in order, falcon tubes arestored −80° C., and the samples are visually rated and image analysis isperformed by Visual Ratings and image analysis.

Visual Ratings: Beginning at 72hrs after processing, each sample will bescored on a 1 (best) to 5 (worst) rating scale against reference photosby multiple individuals independently. It is necessary to ensureadequate lighting to discern differences in browning. Ideally thesamples will be rated at day 3, 5, and 7 according to the referencephotos, and the timing of clamshell evaluations to be done as blocksfrom the field. The combined score for each clamshell will be recorded.

Image Analysis: The lettuce pieces in each black tray will bephotographed at the same time points as the visual ratings. A lightstation with Rosco Litepad HO+, 3″×12″ light bars set at a 40° anglefrom horizontal on either side at a distance of 18″ from the worksurface will be used. Images are collected with a Canon EOS Rebel T3that is positioned at 53 cm above the platform. The amount ofdiscoloration is determined by image analysis using a custom Matlabprogram to quantify the number of brown pixels relative to the number ofpixels representing the total lettuce leaf area.

Example 4 Expression of PPO11 Correlates with Browning and Reduced ShelfLife

The levels of expression of PPO2, PPO8, PPO10, PPO11 and PAL1 weredetermined by testing central mid rib RNA extractions from materialscollected at the time of processing or after 7-9 days of storage inambient air at 4° C. RNA samples were prepared using PureLink® Plant RNAReagent (Ambion) kit following procedures recommended by themanufacturer. QRT-PCRs were performed as described in the operator'smanual using a Stratagene MX3000PTM. Gene-specific PCR primers weredesigned (see Table 2). All amplification plots were analyzed with theMX3000PTM software to obtain threshold cycle (Ct) values. Transcriptabundance was normalized to the transcript abundance of ubiquitin(GenBank accession number EF681766).

TABLE 2 Gene-specific PCR primers: Fluorescent SEQ ID NOs AmpliconPrimer Name Tag 5′→3′ SEQ ID NO: 12 PAL1 PAL1-F — CGTCGAGATTCTGCGAG AAAGSEQ ID NO: 13 PAL1 PAL1-R — GCAAACGTCGTCGATGT AAGC SEQ ID NO: 14 PAL1LsPAL1Probe 6FAM CTCCTCCGTGTTGTTGAT CGTGAATACGTC SEQ ID NO: 15 PPO1PPO1-F — GCAAGTTCAATAAAGCCA TCGA SEQ ID NO: 16 PPO1 PPO1-R —CGCAATAGGCACAATGA ACATT SEQ ID NO: 17 PPO1 LsPPO1Probe 6FAMCCCAGATGACGATCCTC GTAGCTTTAAGC SEQ ID NO: 18 PPO10 PPO10-F —AAGGCTTCCAGAAACAT CAAGAA SEQ ID NO: 19 PPO10 PPO10-R — AGACTCGCCGGAAAAACATCT SEQ ID NO: 20 PPO10 LsPPO10Probe 6FAM CATGCCCATGAACACATA CCCTTTGCASEQ ID NO: 21 PPO11 PPO11-F — GCAAGATCAAGAAGCTC GCTGTA SEQ ID NO: 22PPO11 PPO11-R — ACTCGCCGGAAAAACAT CTTT SEQ ID NO: 23 PPO11 LsPPO11Probe6FAM CGAGCCGATGAACACAT ACCCTTTGC SEQ ID NO: 24 PPO2 PPO2-F —GACGTACCATGGCTAAA AAGCAA SEQ ID NO: 25 PPO2 PPO2-R — CGATGGATTTCCTCGCAACT SEQ ID NO: 26 PPO2 LsPPO2Probe 6FAM CCAGTCCCACGTGCACC CAGGSEQ ID NO: 27 PPO8 PPO8-F — GCTGCAACAGACCCGGT TA SEQ ID NO: 28 PPO8PPO8-R — CTGGCGGGTCCTTCTGA TT SEQ ID NO: 29 PPO8 LsPPO8Probe 6FAMCCGAAGAGGAACAACAC AAAGGACTTCCC SEQ ID NO: 30 Ubiquitin Ubiquitin-F —TTGTCTTGAATTTTAGCT TTGACGTT SEQ ID NO: 31 Ubiquitin Ubiquitin-R —CCTTGACCGGAAAAACA ATCA SEQ ID NO: 32 Ubiquitin LsUbiquitinProbe VICTCAATGGTGTCGGAGCT TTCCACTTCC

A plot of Visual Discoloration vs the gene x time point combinationswith an r>0.7 are shown in FIG. 3a and FIG. 4. PPO11 was weaklyexpressed or not expressed at harvest time but was strongly inducedafter processing and storage. PPO11 induction was observed to have thelargest range of range of expression, as demonstrated in FIG. 5.

The correlation of PPO11 expression and visual discoloration in a largescale experiment in a different environment is shown in FIG. 3B. Again,the PPO11 expression levels 9 days after processing and cold storagewith visual discoloration ratings for two crisphead romaine cross, threeiceberg, and five Romaine cultivars was significant. Each pointrepresents the least square mean for a single cultivar with sixbiological replicates for both gene expression and visual ratings.

In sum, the correlation of expression of PPO11 with a visual browningphenotype was demonstrated in FIGS. 3A-B, and a plot of visualdiscoloration was provided in FIG. 4, demonstrating that browning wasstrongly correlated with expression of PPO11.

Example 5 Marker Selection may be Used to Track the PPO11 Locus

Genetic marker sequences associated with a PPO11 locus with reduced geneexpression may be used to track that locus in a breeding program. APPO11 gene or ortholog thereof gene with reduced expression may beidentified, for example, by screening for naturally occurring lowexpressing lines or by one of the methodologies outlined herein. Once areduced expression PPO11 locus is identified or made, marker sequencesassociated with the PPO11 locus may be used to track the reducedexpression locus in segregating progeny from a plant cross. For example,sequence may be PCR-amplified from sequences flanking PPO11, such as SEQID NO:10 and SEQ ID NO:11 and across the PPO11 genetic region in bothparents of the cross. Single nucleotide polymorphisms or sequencedifferences between the two parents may be compared to identify specificalleles from each parent associated with the normal expression orreduced expression PPO11 gene. Sequence-based assay, (e.g. TaqMan) ofthese single nucleotide polymorphisms or sequence differences in thesegregating progeny of a cross will allow selection of the marker allelelinked to the desirable reduced expression PPO11 gene.

Example 6 Identification of PPO Targets in Potato and Apple

The PPO11 sequence included here may be used to search potato and applegenome sequence data for PPO homologs. Using the methods detailed here,expression data from these PPO genes can be correlated with theincidence of browning to identify PPO homolog targets for increasingshelf life. Once appropriate targets are identified, their reducedexpression may be identified or brought about and tracked via themethodologies described here.

1.-49. (canceled)
 50. A method for identifying a polymorphismgenetically linked to a PPO11 gene, comprising: a) obtaining DNA of apopulation of plants wherein members of the population vary forexpression of PPO11; and b) identifying at least a first polymorphism insaid population that is associated with a reduced expression of PPO11relative to members of the population that do not comprise saidpolymorphism.
 51. (canceled)
 52. A method of identifying a lettuce plantthat displays reduced browning or increased shelf life comprising:detecting in a first lettuce plant at least one polymorphism that isassociated with reduced expression of a PPO11 gene identified by themethod of claim
 50. 53. The method of claim 50, wherein said PPO11 genehas at least 95% sequence identity to SEQ ID NO:9.
 54. The method ofclaim 50, wherein said polymorphism is located in the PPO11 gene. 55.The method of claim 50, wherein said polymorphism is located in a regionflanking the PPO11 gene.
 56. The method of claim 55, wherein the regionflanking the PPO11 gene has at least 95% sequence identity to SEQ IDNO:10.
 57. The method of claim 55, wherein the region flanking the PPO11gene has at least 95% sequence identity to SEQ ID NO:11.